Single domain antibodies to programmed cell death protein 1 (pd-1)

ABSTRACT

The invention relates to PD-1 binding agents that block the interaction of PD-1 with its ligands, and the use of such binding agents in the treatment, prevention and detection of disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 16/475,597, which is a 35 U.S.C. § 371 National Phase Application of International Application No. PCT/GB2018/050036, filed Jan. 8, 2018, which claims the benefit of GB Application Nos. 1700207.2, filed Jan. 6, 2017, 1700208.0, filed Jan. 6, 2017, and 1700210.6, filed Jan. 6, 2017, the content of each is incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing (File Name: 4577_0010007_Seqlisting_ST26.XML, Size: 807,404 bytes, and Date of Creation: Feb. 8, 2023) is hereby incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to PD-1 binding agents, in particular anti-PD-1 V_(H) single domain antibodies (sdAb), and the use of such binding agents in the treatment, prevention and detection of disease.

INTRODUCTION

Antibody-based therapeutics have emerged as important components of therapies for an increasing number of human diseases in such fields as oncology, inflammatory and infectious diseases. Indeed, antibodies are one of the best-selling classes of drugs today; five of the top ten best selling drugs are antibodies.

The Programmed Death 1 (PD-1) protein is encoded by the PDCD1 gene and expressed as a 55 kDa type I transmembrane protein (Agata 1996 Int Immunol 8(5):765-72). PD-1 is an immunoglobulin superfamily member (Ishida 1992 EMBO 11(11):3887-95) and it is an inhibitory member of the extended CD28/CTLA-4 family of T cell regulators. Other members of this family include CD28, CTLA-4, ICOS and BTLA. PD-1 exists as a monomer, lacking the unpaired cysteine residue characteristic of other CD28 family members (Zhang 2004 Immunity 20:337-47). Its cytoplasmic domain contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM) that are phosphorylated during signal transduction (Riley 2009 Immunol Rev 229(1):114-25).

PD-1 is expressed on B cells, T cells, and monocytes (Agata 1996). The role of PD-1 in maintaining immunologic self-tolerance was demonstrated in PDCD1−/− mice, which develop autoimmune disorders (Nishimura 1999 Immunity 11:141-51, Nishimura 2001 Science 291(5502):319-22). The PD-1 pathway therefore regulates antigen responses, balancing autoimmunity and tolerance.

There are two ligands for PD-1 that mediate its regulatory function. PD-L1 (B7-H1) is normally expressed on dendritic cells, macrophages, resting B cells, bone marrow-derived mast cells and T cells as well as non-hematopoietic cell lineages (reviewed in Francisco 2010 Immunol Rev 236:219-42). PD-L2 (B7-DC) is largely expressed on dendritic cells and macrophages (Tseng 2001 J Exp Med 193(7):839-45). Ligand expression is influenced by local mediators and can be upregulated by inflammatory cytokines.

PD-1 is known as an immunoinhibitory protein that negatively regulates TCR signals. The interaction between PD-1 and PD-L1 can act as an immune checkpoint, which can lead to, e.g., a decrease in tumour infiltrating lymphocytes, a decrease in T-cell receptor mediated proliferation, and/or immune evasion by cancerous cells. Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1 or PD-L2; the effect is additive when the interaction of PD-1 with both PD-L1 and PD-L2 is blocked.

As T cells become activated and co-stimulated by antigen-presenting cells (APCs), T cell expression of PD-1 is induced. PD-1 engagement with ligand on the APC cross-links PD-1 and clusters it into the T cell receptor (TCR) complex within the immunological synapse (Yokosuka 2012 J Exp Med 209(9):1201-17). Within the T cell cytoplasm, PD-1 signalling domains ITIM and ITSM are phosphorylated. This induces Src-homology-2 domain-containing tyrosine phosphatase (SHP1/2) that attenuates various components of the T cell receptor (TCR) signalling. T cell activation is dampened, which leads to a reduction in cytokine response, proliferation and cytolytic activity. This downregulation of T cell function serves to prevent overstimulation, tolerising cells against weakly immunogenic self-antigen.

The PD-1 pathway can be exploited in cancer or infection, whereby tumours or viruses can evade effective immune recognition and T cells demonstrate an ‘exhausted’ phenotype. PD-L1 has also been shown to be expressed in many tumour types including urothelial, ovarian, breast, cervical, colon, pancreatic, gastric, melanoma, glioblastoma and non-small cell lung carcinoma (reviewed in Callahan 2014 J Leukoc Biol 94(1):41-53). The cytokines produced by cancer stromal cells can further upregulate PD-L1 in the tumour microenvironment (He 2015 Nature Scientific Reports 5:13110). As a result, tumour-specific T cells become unresponsive through PD-1 signalling and therefore fail to eliminate their target. T regulatory cells (T regs) have also been shown to express high levels of PD-1 and they suppress the anti-tumour response further (Lowther 2016 JCI Insight 1(5):85935).

Disruption of the PD-1:PD-L1 interaction enhances T cell activity. An anti-PD-1 monoclonal antibody demonstrates blockade of the interaction between PD-1 and its ligands (Wang 2014 Cancer Immunol Res 2(9):846-56). T cell function in-vitro can be enhanced by PD-1 blockade, as demonstrated by improved proliferation and cytokine responses in mixed lymphocyte reactions of T cells and dendritic cells. cytotoxic lymphocytes (CTLs) derived from melanoma patients has also been shown to be enhanced by PD-1 blockade in vitro using the antibody OPDIVO (nivolumab), and can become resistant to Treg suppression (Wang 2009 Int Immunol 21(9):1065-1077). This antibody has been tested in clinical dose escalation studies in melanoma, non-small cell lung carcinoma (NSCLC), renal cell cancer (RCC) and others. It shows improved overall survival rates compared to chemotherapy in NSCLC patients. Another PD-1 blocking antibody, KEYTRUDA® (pembrolizumab), demonstrates responses in NSCLC patients refractory to CTLA-4 blockade. OPDIVO® and KEYTRUDA® both functionally block the interaction of human PD-1 with its ligands.

It is possible to induce PD-1 signalling by cross-linking it on the membrane with a combination of anti-PD-1 plus anti-CD3 antibodies (Bennett 2003 J Immunol 170:711-18, Keir 2005 J Immunol 175:7372-7379). This function could be detrimental during an anti-tumour response because T cell activity would be suppressed. If suppression of T cell responses were desired, agonistic anti-PD-1 antibodies or those with effector functions could be used to treat immune-related diseases such as rheumatoid arthritis.

The aim of the present invention is to address the need of alternative antibody-based treatments for use in the treatment of disease, in particular in the treatment of cancer.

SUMMARY OF THE INVENTION

The invention provides isolated single domain antibodies, in particular V_(H) single domain antibodies that bind to PD-1 and that exhibit a number of desirable properties.

The single domain antibodies of the present invention bind to human PD-1 and modulate the interaction of PD-1 with PD-L1 and/or PD-L2. The anti-PD-1 antibodies preferably bind to PD-1 with high affinity and block the functional interaction of PD-1 with PD-L1 and/or PD-L2. In some embodiments, the blocking antibodies of the invention may block the binding of PD-1 to PD-L1, PD-L2 and/or stimulate or enhance T-cell activation. In some embodiments, the blocking antibodies may be useful for stimulating or enhancing the immune response and/or for treating a subject suffering from cancer and other diseases.

Thus, in one aspect, the invention provides isolated single domain antibody that binds to human PD-1 comprising a CDR3 sequence selected from a CDR3 of table 1.

In one embodiment, the single domain antibody comprises a CDR1 as shown in SEQ ID No. 1 or a sequence with at least 80% homology thereto, a CDR2 as shown in SEQ ID No. 2 or a sequence with at least 80% homology thereto and a CDR3 as shown in SEQ ID No. 3 or a sequence with at least 80% homology thereto.

In one embodiment, the isolated single domain antibody comprises CDR1, CDR2 and CDR3 sequences selected from CDR1, CDR2 and CDR3 sequences as shown Table 1.

In one embodiment, the single domain antibody comprises a sequence selected from SEQ ID Nos. 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 272, 276, 280, 284, 288, 292, 369, 373, 377, 381, 385, 389, 393, 397, 401, 405, 409, 413, 417 421, 425, 429, 433, 437, 441, 445, 449, 453, 457, 461, 465, 469, 473, 477, 481, 485, 489, 493, 497, 501, 505, 509, 513, 517, 521, 525, 529 or 533 or a sequence with at least 70%, 80% or 90% homology thereto.

In one embodiment, the single domain antibody is conjugated to a toxin, enzyme, radioisotope, half-life extending moiety, label, therapeutic molecule or other chemical moiety.

In one embodiment, the isolated single domain antibody is obtained or obtainable from a transgenic rodent that expresses a transgene comprising human V, D and J regions.

The invention also provides an isolated binding agent that binds to essentially the same epitope as the single domain antibody described herein.

The invention also provides an isolated binding agent that competes for binding to human PD-1 with the single domain antibody described herein.

The invention also provides an isolated binding agent comprising a single domain antibody described herein.

In one embodiment, said single domain antibody is linked to a second binding molecule that does not bind to PD-1.

In one embodiment, said second single domain antibody binds to an immonooncology target.

In one embodiment, said single domain antibody is linked to a second binding molecule that binds to PD-1.

The invention also provides the use of a single domain antibody described herein in a multispecific or multivalent binding agent.

The invention also provides an immunoconjugate comprising a single domain antibody or a binding agent described herein linked to a therapeutic agent.

The invention also provides a pharmaceutical composition comprising a single domain antibody described herein, a binding agent described herein or an immunoconjugate described herein and a pharmaceutical carrier.

The invention also provides a method for treating a cancer, an immune disorder, neurological disease, inflammatory disorder, allergy, transplant rejection, viral infection, immune deficiency or other immune system-related disorder comprising administering a therapeutically effective amount of a comprising a single domain antibody, an immunoconjugate or a pharmaceutical composition according to claim described herein.

The invention also provides the use of a single domain antibody, a binding agent, an immunoconjugate or a pharmaceutical composition described herein in the manufacture of a medicament for the treatment of a cancer, an immune disorder, neurological disease, inflammatory disorder, allergy, transplant rejection, viral infection, immune deficiency or other immune system-related disorder.

The invention also provides a single domain antibody, a binding agent, an immunoconjugate or a pharmaceutical composition described herein for use as medicament.

The invention also provides single domain antibody, a binding agent, an immunoconjugate or a pharmaceutical composition described herein for use in the treatment of a cancer, an immune disorder, neurological disease, inflammatory disorder, allergy, transplant rejection, viral infection, immune deficiency, and other immune system-related disorder.

The invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a single domain antibody described herein.

The invention also provides a method for producing a single domain antibody described herein comprising expressing a nucleic acid encoding said binding molecule in a host cell and isolating the binding molecule from the host cell.

The invention also provides a kit comprising a single domain antibody, a binding agent, an immunoconjugate or a pharmaceutical composition described herein.

The invention also provides a method for detecting the presence of human PD-1 in a test sample comprising contacting said sample with a single domain antibody described herein and at least one detectable label and detecting binding of said single domain antibody to human PD-1.

The invention also provides a method for producing a single V_(H) domain antibody that binds to human PD-1 comprising

-   -   a) immunising a transgenic animal that expresses a nucleic acid         construct comprising human heavy chain V genes and that is not         capable of making functional endogenous light or heavy chains         with an PD-1 antigen,     -   b) generating a library from said animal     -   c) isolating single V_(H) domain antibodies from said libraries         and     -   d) identifying a single V_(H) domain antibody that binds to         human PD-1 and     -   e) isolating said antibody.

The invention also provides a single V_(H) domain antibody obtained or obtainable by the method above.

The invention also provides an isolated heavy chain only antibody comprising a V_(H) domain that binds to human PD-1.

The invention also provides a heavy chain only antibody comprising a V_(H) domain that binds to human PD-1 obtained or obtainable from a transgenic mouse which expresses human V, D and J loci and does not produce functional endogenous lambda and kappa light chains and heavy chain.

The invention also provides bispecific molecule comprising the single domain antibody described above linked to a second functional moiety having a different binding specificity than said single domain antibody.

Another aspect of the invention pertains to methods of modulating immune responses using anti-PD-1 single domain antibodies described herein.

The invention further provides a method of inhibiting growth of tumor cells in vivo using anti-PD-1 single domain antibodies described herein.

FIGURES

The invention is further described in the following non-limiting figures.

FIGS. 1A-1B: FIG. 1A) Binding Assay: V_(H) were identified that bound to the CHO human PD-1 and FIG. 1B) Inhibition Assay: Inhibition of PD-1 binding to PD-L1.

FIG. 2 : PD-1/PD-L2 inhibition assay using different single domain antibodies.

FIGS. 3A-3C: Species cross reactivity testing. FIG. 3A) V_(H) single domain antibody 1.1 and 1.57 binding to human PD-1; FIG. 3B) V_(H) single domain antibody 1.1 and 1.57 binding to cynomolgus PD-1 recombinant protein; FIG. 3C) V_(H) single domain antibody 1.1 and 1.57 binding to mouse PD-1 protein.

FIGS. 4A-4C: FMAT Inhibition assay using V_(H) single domain antibodies. FIG. 4A) CHO PD-1/PD-L1 assay with monovalent V_(H) single domain antibodies; FIG. 4B) CHO PD-1/PD-L2 assay with monovalent V_(H) single domain antibodies; FIG. 4C) CHO PD-1/PD-L1 assay with bivalent agents.

FIGS. 5A-5C: Serum stability of different V_(H) single domain antibodies. FIG. 5A) serum stability of 1.57 V_(H) single domain antibodies; FIG. 5B) serum stability of 1.64 V_(H) single domain antibodies FIG. 5C) serum stability of formatted V_(H) 1.57-4GS-MSA binder

FIG. 6 : Binding of V_(H) single domain antibody to CD4 T-cells.

FIGS. 7A-7B: shows a co-culture of dendritic cells and CD4+ T cells.: FIG. 7A) Effects of PD-1 specific Humabody® 1.57 on human T cell activation in a Mixed Lymphocyte Reaction; FIG. 7B) Effects of PD-1 specific Humabody® 1.92 on human T cell activation in a Mixed Lymphocyte Reaction.

FIG. 8 : In Vivo Efficacy of Humabody® Compounds in HuGEMM PD-1 Model with Subcutaneous MC38 Mouse Colon Adenocarcinoma.

FIG. 9 : Monovalent V_(H) single domain antibody and biparatopic in the functional reporter assay.

DETAILED DESCRIPTION

The various aspects and embodiments will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012); Therapeutic Monoclonal Antibodies: From Bench to Clinic, Zhiqiang An (Editor), Wiley, (2009); and Antibody Engineering, 2nd Ed., Vols 1 and 2, Ontermann and Dubel, eds., Springer-Verlag, Heidelberg (2010).

Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

The invention thus provides isolated single domain antibodies that bind human PD-1 and block the interaction of PD-1 with PD-L1 and/or the interaction of PD-1 with PD-L2, pharmaceutical compositions comprising such binding molecules, as well as isolated nucleic acids, isolated recombinant expression vectors and isolated host cells for making such binding proteins. Also provided are methods of using the single domain antibodies disclosed herein to detect human PD-1 and methods of treating disease. In another aspect, the invention provides binding molecules comprising a single domain antibody that binds human PD-1 and block the interaction of PD-1 with PD-L1 and/or the interaction of PD-1 with PD-L2 as described herein.

In one aspect, the invention relates to a single domain antibody, wherein the single domain antibody exhibits one or more of the following properties:

-   -   (a) binds to human PD-1 with a KD as measured in the examples;     -   (b) increases IL-2 secretion in an MLR assay;     -   (c) binds to human PD-1 and cynomolgus monkey PD-1;     -   (d) does not bind to mouse PD-1;     -   (e) inhibits the binding of PD-L1 and/or PD-L2 to PD-1;     -   (f) inhibits tumor cell growth in vivo (e.g. as measured in the         examples).

Suitable assays to measure the properties as set out above are described in the examples.

In preferred embodiments, the single domain antibody is a single domain antibody wherein the domain is a human variable heavy chain (V_(H)) domain. Thus, in certain embodiments, the invention provides isolated single domain antibodies that bind human PD-1, wherein the domain is a variable heavy chain domain, preferably a V_(H) domain and wherein said single domain antibodies bind to human PD-1 and block the interaction of PD-1 with PD-L1 and/or the interaction of PD-1 with PD-L2.

The properties of the single domain antibodies of the invention as described above can be exploited in therapeutic methods and uses as described herein.

In particular, as explained below, the single domain antibodies of the invention can be used in a multivalent or multispecific format. Thus, the invention also relates to multifunctional binding agents comprising a single domain antibody as described herein.

Molecules of the invention bind specifically to wild type human PD-1 (UniProt Accession No. Q15116, GenBank Accession No. U64863, SEQ ID No. 576). Residues 1-20 correspond to the pre-sequence, residues 171 and beyond make up the transmembrane helix and the intracellular domain of PD-1.

Unless otherwise specified, the term PD-1 as used herein refers to human PD-1. The terms “Programmed Death 1,” “Programmed Cell Death 1,” “Protein PD-1,” “PD-1,” PD1,” “PDCD1,” “hPD-1” and “hPD-1” are used interchangeably, and include variants, isoforms, species homologs of human PD-1.

The terms “PD-1 binding molecule/protein/polypeptide/agent”, “PD-1 antigen binding molecule protein/polypeptide/agent”, “anti-PD-1 single domain antibody”, “anti-PD-1 single immunoglobulin variable domain”, “anti-PD1 heavy chain only antibody” or “anti-PD-1 antibody” all refer to a molecule capable of specifically binding to the human PD-1 antigen. The binding reaction may be shown by standard methods, for example with reference to a negative control test using an antibody of unrelated specificity. The term “PD-1 binding molecule/agent” includes a PD-1 binding protein.

An antibody or binding molecule of the invention, including a single domain antibody and multivalent or multispecific binding agent described herein, “which binds” or is “capable of binding” an antigen of interest, e.g. PD-1, is one that binds the antigen with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting a cell or tissue expressing the antigen.

Binding molecules of the invention, including the single domain antibodies and multivalent or multispecific binding agents described herein, bind specifically to human PD-1. In other words, binding to the PD-1 antigen is measurably different from a non-specific interaction. As demonstrated in the examples, the single domain antibodies of the invention do not cross react with mouse PD-1. Preferably, the single domain antibodies of the invention bind to human PD-1 and also bind to cyno PD-1.

The term “specific binding” or “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a KD for the target of at least about 10⁻⁴ M, alternatively at least about 10⁻⁵ M, alternatively at least about 10⁻⁸ M, alternatively at least about 10⁻⁷ M, alternatively at least about 10⁻⁸ M, alternatively at least about 10⁻⁹ M, alternatively at least about 10⁻¹⁰ M, alternatively at least about 10⁻¹¹ M, alternatively at least about 10⁻¹² M, or greater. In one embodiment, the term “specific binding” refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

The term “antibody” broadly refers to any immunoglobulin (Ig) molecule, or antigen binding portion thereof, comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art.

In a full-length antibody, each heavy chain is comprised of a heavy chain variable region or domain (abbreviated herein as HCVR) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, C_(H)1, C_(H)2 and C_(H)3. Each light chain is comprised of a light chain variable region or domain (abbreviated herein as LCVR) and a light chain constant region. The light chain constant region is comprised of one domain, C_(L).

The heavy chain and light chain variable regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each heavy chain and light chain variable region is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG2, IgG 3, IgG4, IgA1 and IgA2) or subclass.

The term “CDR” refers to the complementarity-determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1, CDR2 and CDR3, for each of the variable regions. The term “CDR set” refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs can be defined differently according to different systems known in the art.

The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain).

The system described by Kabat is used herein unless otherwise specified. The terms “Kabat numbering”, “Kabat definitions” and “Kabat labeling” are used interchangeably herein. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion.

A chimeric antibody is a recombinant protein that contains the variable domains including the complementarity determining regions (CDRs) of an antibody derived from one species, preferably a rodent antibody, while the constant domains of the antibody molecule are derived from those of a human antibody. For veterinary applications, the constant domains of the chimeric antibody may be derived from that of other species, such as a cat or dog.

A humanized antibody is a recombinant protein in which the CDRs from an antibody from one species; e.g., a rodent antibody, are transferred from the heavy and light variable chains of the rodent antibody into human heavy and light variable domains (e.g., framework region sequences). The constant domains of the antibody molecule are derived from those of a human antibody. In certain embodiments, a limited number of framework region amino acid residues from the parent (rodent) antibody may be substituted into the human antibody framework region sequences.

The term “antigen binding site” refers to the part of the antibody or antibody fragment that comprises the area that specifically binds to an antigen. An antigen binding site may be provided by one or more antibody variable domains. Preferably, an antigen binding site is comprised within the associated V_(H) and V_(L) of an antibody or antibody fragment.

An antibody fragment is a portion of an antibody, for example as F(ab′)₂, Fab, Fv, sFv and the like. Functional fragments of a full length antibody retain the target specificity of a full length antibody. Recombinant functional antibody fragments, such as Fab (Fragment, antibody), scFv (single chain variable chain fragments) and single domain antibodies (dAbs) have therefore been used to develop therapeutics as an alternative to therapeutics based on mAbs.

scFv fragments (˜25 kDa) consist of the two variable domains, V_(H) and V_(L). Naturally, V_(H) and V_(L) domain are non-covalently associated via hydrophobic interaction and tend to dissociate. However, stable fragments can be engineered by linking the domains with a hydrophilic flexible linker to create a single chain Fv (scFv).

The smallest antigen binding fragment is the single variable fragment, namely the V_(H) or V_(L) domain. Binding to a light chain/heavy chain partner respectively is not required for target binding. Such fragments are used in single domain antibodies. A single domain antibody (˜12 to 15 kDa) therefore has either the V_(H) or V_(L) domain.

In one aspect, the invention relates to an isolated single domain antibody, an isolated variable single domain or an isolated immunoglobulin single variable domain wherein said isolated single domain antibody, isolated variable single domain or isolated immunoglobulin single variable domain binds to human PD-1 and blocks the interaction of PD-1 and PD-L1 or PD-L2.

The terms “single domain antibody, variable single domain or immunoglobulin single variable domain (ISV)” are all well known in the art and describe the single variable fragment of an antibody that binds to a target antigen. These terms are used interchangeably herein. As explained below, preferred embodiments of the various aspects of the invention relate to single heavy chain variable domain antibodies/immunoglobulin heavy chain single variable domains which bind a PD-1 antigen in the absence of light chain. Human heavy chain single variable domain antibodies are particularly preferred. Such binding molecules are also termed Humabody® herein. Humabody® is a registered trademark of Crescendo Biologics Ltd.

Thus, in some preferred embodiments, the isolated binding agents/molecules of the invention comprise or consist of at least one single domain antibody wherein said domain is a human heavy chain variable domain. Thus, in one aspect, the binding agents of the invention comprise or consist of at least one human immunoglobulin single variable heavy chain domain; they are devoid of V_(L) domains.

The term “isolated” single domain antibody refers to a single domain antibody that is substantially free of other single domain antibodies, antibodies or antibody fragments having different antigenic specificities. Moreover, an isolated single domain antibody may be substantially free of other cellular material and/or chemicals.

A “blocking single domain antibody or antibody” or a “neutralizing single domain antibody or antibody”, as used herein refers to an antibody whose binding to PD-1 results in inhibition of at least one biological activity of PD-1. For example, a single domain antibody of the invention may prevent or block PD-1 binding to PD-L1 and/or PD-L2. In one embodiment, the single domain antibody of the invention blocks PD-1 binding to PD-L1. In one embodiment, the single domain antibody of the invention blocks PD-1 binding to PD-L2.

Each single V_(H) domain antibody comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Thus, in one embodiment of the invention, the domain is a human variable heavy chain (V_(H)) domain with the following formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

Modifications to the C or N-terminal V_(H) framework sequence may be made to the single domain antibodies of the invention to improve their properties. For example, the V_(H) domain may comprise C or N-terminal extensions or deletions. C-terminal extensions can be added to the C-terminal end of a V_(H) domain which terminates with the residues VTVSS (SEQ ID No. 585).

In one embodiment, the single domain antibodies of the invention comprise C-terminal extensions or deletions of from 1 to 50 residues, for example 1 to 25, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acids. In one embodiment, the single domain antibodies of the invention comprise additional amino acids of the human C_(H)1 domain thus that the C terminal end extends into the C_(H)1 domain. In one embodiment, said extension comprises at least one alanine residue, for example a single alanine residue, a pair of alanine residues or a triplet of alanine residues.

Additional C or N-terminal residues can be linkers that are used to conjugate the single domain antibodies of the invention to another moiety, or tags that aid the detection of the molecule. Such tags are well known in the art and include for, example linker His tags, e.g., hexa-His (HHHHHH, SEQ ID No. 586) or myc tags.

As used herein, the term “homology” generally refers to the percentage of amino acid residues in a sequence that are identical with the residues of the reference polypeptide with which it is compared, after aligning the sequences and in some embodiments after introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Thus, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. Neither N- or C-terminal extensions, tags or insertions shall be construed as reducing identity or homology. Methods and computer programs for the alignment are well known. The percent identity between two amino acid sequences can be determined using well known mathematical algorithms.

According to the various aspects and embodiments of the invention, the variable domain of the single domain antibodies of the invention is preferably a human variable domain (V_(H)). As used herein, a human V_(H) domain includes a fully human or substantially fully human V_(H) domain. As used herein, the term human V_(H) domain also includes V_(H) domains that are isolated from heavy chain only antibodies made by transgenic mice expressing fully human immunoglobulin heavy chain loci, in particular in response to an immunisation with an antigen of interest, for example as described in WO2016/062990 and in the examples. In one embodiment, a human V_(H) domain can also include a V_(H) domain that is derived from or based on a human V_(H) domain amino acid or nucleic acid sequence encoding such V_(H) domain. Thus, the term includes variable heavy chain regions derived from or encoded by human germline immunoglobulin sequences. A substantially human V_(H) domain or V_(H) domain that is derived from or based on a human V_(H) domain may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced in vitro, e.g. by random or site-specific mutagenesis, or introduced by somatic mutation in vivo). The term “human V_(H) domain” therefore also includes a substantially human V_(H) domain wherein one or more amino acid residue has been modified. For example, a substantially human V_(H) domain the V_(H) domain may include up to 10, for example 1, 2, 3, 4 or 5 amino acid modifications compared to a fully human sequence.

However, the term “human V_(H) domain” or “substantially human V_(H) domain”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Preferably, the term “human V_(H) domain”, as used herein, is also not intended to include camelized V_(H) domains, that is human V_(H) domains that have been specifically modified, for example in vitro by conventional mutagenesis methods to select predetermined positions in the V_(H) domains sequence and introduce one or more point mutation at the predetermined position to change one or more predetermined residue to a specific residue that can be found in a camelid V_(HH) domain.

In one embodiment, the invention relates to an isolated single V_(H) domain antibody that binds to human PD-1 comprising a CDR3 sequence as shown Table 1 below or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence identity thereto. For example, the single V_(H) domain antibody capable of binding to human PD-1 may comprise a CDR3 sequence selected from SEQ ID Nos. 3 or 441 or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence homology to one of these sequences. In one embodiment, said sequence homology is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In one embodiment, said sequence homology is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

In one embodiment, the V_(H) single domain antibody has a CDR1 as shown in SEQ ID No. 1 or SEQ ID No. 1 with 1 or 2 amino acid substitutions, a CDR2 as shown in SEQ ID No. 2 or SEQ ID No. 2 with 1 to 5 amino acid substitutions and a CDR3 as shown in SEQ ID No. 3 or SEQ ID No. 3 with 1 to 5 amino acid substitutions.

In one embodiment, the V_(H) single domain antibody has a CDR3 sequence comprising SEQ ID No. 3 or a sequence having at least 70%, at least 80%, at least 90%, or at least 95% homology to SEQ ID No. 3. In one embodiment, said sequence homology is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

In one embodiment, the V_(H) single domain antibody comprises a combination of CDR1, 2 and 3 sequences selected from the CDR1, 2 and 3 sequences in Table 1 or combinations thereof. In one embodiment, the V_(H) single domain antibody comprises a set of CDR1, 2 and 3 sequences selected from the sets of CDR1, 2 and 3 sequences as shown for the any of the clones in Table 1. Thus, in one aspect, the isolated single domain antibody comprises a CDR1, CDR2 and CDR3 selected from CDRs1-3 of full length sequences SEQ ID Nos: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 272, 276, 280, 284, 288, 292, 369, 373, 377, 381, 385, 389, 393, 397, 401, 405, 409, 413, 417 421, 425, 429, 433, 437, 441, 445, 449, 453, 457, 461, 465, 469, 473, 477, 481, 485, 489, 493, 497, 501, 505, 509, 513, 517, 521, 525, 529 or 533.

Accordingly, in one embodiment, the V_(H) single domain antibody comprises CDR1 having SEQ ID No. 1, CDR2 having SEQ ID No. 2 and CDR3 having SEQ ID No. 3 (CDRs of SEQ ID NO. 4), a CDR1 having SEQ ID No. 5, CDR2 having SEQ ID No. 6 and CDR3 having SEQ ID No. 7 (CDRs of SEQ ID NO. 8) and so forth. Thus, the V_(H) single domain antibody comprises one of the following CDR combinations: SEQ ID Nos. 1, 2, 3; SEQ ID Nos. 5, 6, 7; SEQ ID Nos. 9, 10, 11; SEQ ID Nos. 13, 14, 15; SEQ ID Nos. 17, 18, 19; SEQ ID Nos. 21, 22, 23; SEQ ID Nos. 25, 26, 27; SEQ ID Nos. 29, 30, 31; SEQ ID Nos. 33, 34, 35; SEQ ID Nos. 37, 38, 39; SEQ ID Nos. 41, 42, 43, SEQ ID Nos. 45, 46, 47; SEQ ID Nos. 49, 50, 51; SEQ ID Nos. 53, 54, 55; SEQ ID Nos. 57, 58, 59; SEQ ID Nos. 61, 62, 63; SEQ ID Nos.65, 66, 67; SEQ ID Nos. 69, 70, 71; SEQ ID Nos.73, 74, 75; SEQ ID Nos. 77, 78, 79; SEQ ID Nos. 101, 102, 103; SEQ ID Nos. 105, 106, 107; SEQ ID Nos. 109, 110, 111; SEQ ID Nos. 113, 114, 115; SEQ ID Nos. 117, 118, 119; SEQ ID Nos. 121, 122, 123; SEQ ID Nos. 125, 126, 127; SEQ ID Nos. 129; 130; 131; SEQ ID Nos. 133, 134, 15; SEQ ID Nos. 137, 18, 139; SEQ ID Nos. 141, 142, 143; SEQ ID Nos. 145, 146, 147; SEQ ID Nos. 149, 150, 151; SEQ ID Nos. 153, 154, 155; SEQ ID Nos. 157, 158, 159; SEQ ID Nos. 161, 162, 163; SEQ ID Nos. 165, 166, 167; SEQ ID Nos. 169, 170, 171; SEQ ID Nos. 173, 174, 175; SEQ ID Nos. 177, 178, 179; SEQ ID Nos. 181, 182, 183; SEQ ID Nos. 185, 186, 187; SEQ ID Nos. 189, 190, 191; SEQ ID Nos. 193, 194, 195; SEQ ID Nos. 197, 198, 199; SEQ ID Nos. 201, 202, 203; SEQ ID Nos. 205, 206, 207; SEQ ID Nos. 209, 210, 211; SEQ ID Nos. 213, 214, 215; SEQ ID Nos. 217, 218, 219, SEQ ID Nos. 221, 222, 223, SEQ ID Nos. 225, 226, 227; SEQ ID Nos. 229, 230, 231; SEQ ID Nos. 233, 234, 235; SEQ ID Nos. 237, 238, 239; SEQ ID Nos. 241, 242, 243; SEQ ID Nos. 245, 246, 247; SEQ ID Nos. 249, 250, 251; SEQ ID Nos. 253, 254, 255; SEQ ID Nos. 257, 258, 259; SEQ ID Nos. 261, 262, 263; SEQ ID Nos. 265, 266, 267; SEQ ID Nos. 269, 270, 271; SEQ ID Nos. 273, 274, 275; SEQ ID Nos. 277, 278, 279; SEQ ID Nos. 281, 282, 283; SEQ ID Nos. 285, 286, 287; SEQ ID Nos. 289, 290, 291; SEQ ID Nos. 366, 367, 368; SEQ ID Nos. 370, 371, 372; SEQ ID Nos. 374, 375, 376; SEQ ID Nos. 378, 379, 380; SEQ ID Nos. 382, 383, 384; SEQ ID Nos. 386, 387, 388; SEQ ID Nos. 390, 391, 392; SEQ ID Nos. 394, 395, 396; SEQ ID Nos. 398, 399, 400; SEQ ID Nos. 403, 403, 404; SEQ ID Nos. 406, 407, 408; SEQ ID Nos. 410, 411, 412; SEQ ID Nos. 414, 415, 416; SEQ ID Nos. 418, 419, 420; SEQ ID Nos. 422, 423, 425; SEQ ID Nos. 426, 427, 428; SEQ ID Nos. 430, 431, 432, SEQ ID Nos. 434, 436, 436; SEQ ID Nos. 438, 439, 440; SEQ ID Nos. 442, 443, 444; SEQ ID Nos. 446, 447, 448; SEQ ID Nos. 450, 451, 452; SEQ ID Nos. 454, 455, 456; SEQ ID Nos. 458, 459, 460; SEQ ID Nos. 462, 463, 464; SEQ ID Nos. 466, 467, 468; SEQ ID Nos. 470, 471, 472, SEQ ID Nos.474, 475, 476; SEQ ID Nos. 478, 479, 480; SEQ ID Nos. 482, 483, 484; SEQ ID Nos. 486, 487, 488; SEQ ID Nos. 490, 491, 492; SEQ ID Nos. 494, 495, 496; SEQ ID Nos. 498, 499, 500; SEQ ID Nos. 502, 503, 504; SEQ ID Nos. 506, 507, 508; SEQ ID Nos. 510, 511, 512; SEQ ID Nos. 514, 515, 516; SEQ ID Nos. 518, 519, 520; SEQ ID Nos. 522, 523, 524; SEQ ID Nos. 526, 527, 528; SEQ ID Nos. 530, 531, 532. In one embodiment, the CDRs are SEQ ID NO. 438, 439, 440.

In another embodiment, said CDR1 comprises or consists of the amino acid sequence SEQ ID NO. 1 or a sequence with at least at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% homology thereto. In one embodiment, said CDR2 comprises or consists of the amino acid sequence SEQ ID No. 2 or a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% homology thereto. In one embodiment, said CDR3 comprises or consists of the amino acid sequence SEQ ID No. 3 or a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% homology thereto.

In another embodiment, the V_(H) single domain antibody comprises or consists of a polypeptide sequence as shown for any one of V_(H) single domain antibodies 1.1 to 1.115 in Table 1 or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence homology thereto. In one embodiment, the invention relates to a V_(H) single domain antibody which has combinations of CDR1, CDR2 and CDR3 as shown for any of V_(H) single domain antibody 1.41 to 1.115. In one embodiment, the invention relates to a V_(H) single domain antibody which has a CDR1, CDR2 and CDR3 sequence as shown for V_(H) single domain antibody 1.57. In one embodiment, the invention relates to a V_(H) single domain antibody which has combinations of CDR1, CDR2 and CDR3 as shown for any of V_(H) single domain antibody 1.62 to 1.73. In one embodiment, the invention relates to a V_(H) single domain antibody which has combinations of CDR1, CDR2 and CDR3 as shown for any of V_(H) single domain antibody 1.74 to 1.115. In one embodiment, the invention relates to a V_(H) single domain antibody which has combinations of CDR1, CDR2 and CDR3 as for V_(H) single domain antibody 1.92.

In one embodiment, the V_(H) single domain antibody comprises or consists of an amino acid sequence selected from SEQ ID Nos. 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 272, 276, 280, 284, 288, 292, 369, 373, 377, 381, 385, 389, 393, 397, 401, 405, 409, 413, 417 421, 425, 429, 433, 437, 441, 445, 449, 453, 457, 461, 465, 469, 473, 477, 481, 485, 489, 493, 497, 501, 505, 509, 513, 517, 521, 525, 529 or 533 or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence homology thereto. In one embodiment, the V_(H) single domain antibody comprises or consists of a sequence selected from SEQ ID Nos. 4, 228, 232, 236, 240, 244, 248, 252, 256, 260, 369, 373, 377, 381, 385, 389, 393, 397, 401, 405, 409, 413, 417 421, 425, 429, 433, 437, 441, 445, 449, 453, 457, 461, 465, 469, 473, 477, 481, 485, 489, 493, 497, 501, 505, 509, 513, 517, 521, 525, 529 or 533 or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence homology thereto. In one embodiment, the V_(H) single domain antibody comprises or consists of a sequence selected from SEQ ID No. 4, 228, 381, 176, 381,441 or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence homology thereto. In one embodiment, said sequence homology is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In one embodiment, the V_(H) single domain antibody comprises or consists of SEQ ID No. 441.

TABLE 1 Full length sequences and CDR sequences of V_(H) single domain antibodies Name CDR1 CDR2 CDR3 Full length 1.1 DYTMT YISTGGTIKY EAPLRLGESPHD SEQ NO. 4 SEQ YTDSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYTMT NO. 1 SEQ NO. 2 SEQ NO. 3 WMRQAPGKGLEWVSYISTGGTIKYYTDSVKGRFTIS RDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGES PHDAFDIWGQGTMVTVSS 1.2 DYTMT YISTGGSIKY EAPLRLGESPHD SEQ NO. 8 SEQ YTDSVKG AFDI EVQLLESGGGLVKPGGSLRLSCAASGFTFSDYTMTW NO. 5 SEQ NO. 6 SEQ NO. 7 MRQAPGKGLEWVSYISTGGSIKYYTDSVKGRFTISRD NAKNSLYLQMNSLRADDTAVYYCAREAPLRLGESPH DAFDIWGQGTMVTVSS 1.3 DYTMT YISTGGSIKY EAPLRLGESPHD SEQ NO. 12 SEQ YTDSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYTMT NO. 9 SEQ NO. 10 SEQ NO. 11 WMRQAPGKGLEWVSYISTGGSIKYYTDSVKGRFTIS RDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGES PHDAFDIWGQGTMVTVSS 1.4 DYYMI YISGGGTTK EAPLRLGETPHD SEQ NO. 16 SEQ YYTDSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMIW NO. 13 SEQ NO. 14 SEQ NO. 15 MRQAPGKGLEWVSYISGGGTTKYYTDSVKGRFTISR DNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGETP HDAFDIWGQGTMVTVSS 1.5 DYTMT YISTGGNTK EAPLRLGESPHD SEQ NO. 20 SEQ YYTDSVKG AFDI EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYTMTW NO. 17 SEQ NO. 18 SEQ NO. 19 MRQAPGKGLEWVSYISTGGNTKYYTDSVKGRFTISR DNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGESP HDAFDIWGQGTMVTVSS 1.6 DYTMT YISTGGTIKY EAPLRLGESPHD SEQ NO. 24 SEQ YTDSVKG AFDI EVQLLESGGGLVKPGGSLRLSCAASGFTFSDYTMTW NO. 21 SEQ NO. 22 SEQ NO. 23 MRQAPGKGLEWVSYISTGGTIKYYTDSVKGRFTISRD NAKNSLYLQMSSLRADDTAVYYCAREAPLRLGESPH DAFDIWGQGTMVTVSS 1.7 DYTMT YISTGGTIKY EAPLRLGESPHD SEQ NO. 28 SEQ YTDSVKG AFDI EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYTMTW NO. 25 SEQ NO. 26 SEQ NO. 27 MRQAPGKGLEWVSYISTGGTIKYYTDSVKGRFTISRD NAKNSLYLQMNSLRADDTAVYYCAREAPLRLGESPH DAFDIWGQGTMVTVSS 1.8 DYTMT YISTGGSIKY EAPLRLGESPHD SEQ NO. 32 SEQ YTDSVKG AFDI EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYTMTW NO. 29 SEQ NO. 30 SEQ NO. 31 MRQAPGKGLEWVSYISTGGSIKYYTDSVKGRFTISRD NAKNSLYLQMNSLRAGDTAVYYCAREAPLRLGESPH DAFDIWGQGTMVTVSS 1.9 DYTMT YISTGGTIKY EAPLRLGESPHD SEQ NO. 36 SEQ YTDSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFGDYTMT NO. 33 SEQ NO. 34 SEQ NO. 35 WMRQAPGKGLEWVSYISTGGTIKYYTDSVKGRFTIS RDNAKNSLYLQMDSLRADDTAVYYCAREAPLRLGES PHDAFDIWGQGTMVTVSS 1.10 DYTMS YISLGGNTK EAPLRLGESPHD SEQ NO. 40 SEQ YYTDSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYTMS NO. 37 SEQ NO. 38 SEQ NO. 39 WMRQAPGKGLEWISYISLGGNTKYYTDSVKGRFTIS RDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGES PHDAFDIWGQGTMVTVSS 1.11 DYDMT YISRGGSTK EAPLRLGETPHD SEQ NO. 44 SEQ YYADSVKG AFDI EVQLLESGGGLVKPGGSLRLSCAASGFTFSDYDMTW NO. 41 SEQ NO. 42 SEQ NO. 43 IRQAPGKGQEWVSYISRGGSTKYYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGETPH DAFDIWGQGTMVTVSS 1.12 DYYMG YISSSGSTIY EAPLRLGESPHD SEQ NO. 48 SEQ YADSVKG AFDI EVQLLESGGGVVKPGGSLRLSCAASGFTFSDYYMG NO. 45 SEQ NO. 46 SEQ NO. 47 WIRQAPGKGLEWISYISSSGSTIYYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGESPH DAFDIWGQGTMVTVSS 1.13 DYTMT YISTGGTIKY EAPLRLGESPHD SEQ NO. 52 SEQ YTDSVKG AFDI EVQLLESGGGLVKPGGSLRLSCAASGFTFSDYTMTW NO. 49 SEQ NO. 50 SEQ NO. 51 MRQAPGKGLEWVSYISTGGTIKYYTDSVKGRFTISRD NAKNSLYLQMNSLRADDTAVYYCAREAPLRLGESPH DAFDIWGQGTMVTVSS 1.14 DYTMT YISTGGTIKY EAPLRLGESPHD SEQ NO. 56 SEQ YTDSVKG AFDI EVQLVESGGGLVQPGRSLRLSCAASGFTFSDYTMT NO. 53 SEQ NO. 54 SEQ NO. 55 WMRQAPGKGLEWVSYISTGGTIKYYTDSVKGRFTIS RDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGES PHDAFDIWGQGTMVTVSS 1.15 DNSMS YISSSGSTIY EAPLRLGESPHD SEQ NO. 60 SEQ YADSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDNSMS NO. 57 SEQ NO. 58 SEQ NO. 59 WIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISR DNAKNSLYLQMNTLRAEDTAVYYCAKEAPLRLGESP HDAFDIWGQGTMVTVSS 1.16 DYTMS YISTGGSIKY EAPLRLGESPHD SEQ NO. 64 SEQ YTDSVKG AFDI EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYTMSW NO. 61 SEQ NO. 62 SEQ NO. 63 MRQAPGKGLEWVSYISTGGSIKYYTDSVKGRFTISRD NAKNSLYLQMNSLRADDTAVYYCAREAPLRLGESPH DAFDIWGQGTMVTVSS 1.17 DYTMT YISTGGSIKY EAPLRLGESPHD SEQ NO. 68 SEQ YTDSVKG AFDI EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYTMTW NO. 65 SEQ NO. 66 SEQ NO. 67 MRQAPGKGLEWVSYISTGGSIKYYTDSVKGRFTISRD NAKNSLYLQMNSLRVDDTAVYYCAREAPLRLGESPH DAFDIWGQGTMVTVSS 1.18 DYTMT YISTGGTIKY EAPLRLGESPHD SEQ NO. 72 SEQ YTDSVKG AFDI QVQLLESGGGLVKPGGSLRLSCAASGFTFSDYTMT NO. 69 SEQ NO. 70 SEQ NO. 71 WMRQAPGKGLEWVSYISTGGTIKYYTDSVKGRFTIS RDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGES PHDAFDIWGQGTMVTVSS 1.19 DYTMT YISTGGSIKY EAPLRLGETPHD SEQ NO. 76 SEQ YTDSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYTMT NO. 73 SEQ NO. 74 SEQ NO. 75 WMRQAPGKGLEWVSYISTGGSIKYYTDSVKGRFTIS RDNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGET PHDAFDIWGQGTMVTVSS 1.20 DYTMT YISSGGSIKF EAPLRLGESPHD SEQ NO. 80 SEQ YADSVKG AFDI EVQLLESGGGLVKPGGSLRLSCAASGFTFSDYTMTW NO. 77 SEQ NO. 78 SEQ NO. 79 MRQAPGKGLEWVSYISSGGSIKFYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGESPH DAFDIWGQGTMVTVSS 1.21 DYTMT YISTGGSIKY EAPLRLGESPHD SEQ NO. 84 SEQ YTDSVKG AFDT EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYTMTW NO. 81 SEQ NO. 82 SEQ NO. 83 MRQAPGKGLEWVSYISTGGSIKYYTDSVKGRFTISRD NAKNSLYLQMNSLRADDTAVYYCAREAPLRLGESPH DAFDTWGQGTMVTVSS 1.22 DYTMT YISTGGSIKY EAPLRLGESPHD SEQ NO. 88 SEQ YTDSVKG AFDI EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYTMTW NO. 85 SEQ NO. 86 SEQ NO. 87 MRQAPGKGLEWVSYISTGGSIKYYTDSVKGRFTISRD NAKNSLYLQMNSLRADDTAVYYCAREAPLRLGESPH DAFDIWGQGTMVTVSS 1.23 DYTMS YISTGGTIKY EAPLRLGESPHD SEQ NO. 92 SEQ YTDSVKG AFDI QITLKESGGGLVKPGGSLRLSCAASGFTFSDYTMSW NO. 89 SEQ NO. 90 SEQ NO. 91 MRQAPGKGLEWVSYISTGGTIKYYTDSVKGRFTISRD NAKNSLYLQMNSLRADDTAVYYCAREAPLRLGESPH DAFDIWGQGTMVTVSS 1.24 DYDMY YISRGGSVT EAPLRLGETPHA SEQ NO. 96 SEQ YYADSVKG AFDI QVTLKESGGGLVKPGGSLRLSCAASGFTFSDYDMY NO. 93 SEQ NO. 94 SEQ NO. 95 WIRQAPGKGLEWVSYISRGGSVTYYADSVKGRFTIS RDNAKNALYLQMNSLRAEDMAVYFCATEAPLRLGE TPHAAFDIWGQGTMVTVSS 1.25 DYYMS FISSSGSTTY EAPLRLGESPHD SEQ NO. 100 SEQ YADSVKG AFDF QVTLKESGGGLVKPGGSLRLSCAASGFTFSDYYMSW NO. 97 SEQ NO. 98 SEQ NO. 99 FRQAPGKEREWISFISSSGSTTYYADSVKGRFTISRDN AKNSLYLQMNSLRAEDTAVYYCAREAPLRLGESPHD AFDFWGQGTMVTVSS 1.26 DNSMS YISSSGSTIY EAPLRLGESPHD SEQ NO. 104 SEQ YADSVKG AFDI QVTLKESGGGLVKPGGSLRLSCAASGFTFSDNSMS NO.  SEQ NO.  SEQ NO. 103 WIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISR 101 102 DNAKNSLYLQMNTLRAEDTAVYYCAKEAPLRLGESP HDAFDIWGQGTMVTVSS 1.27 DYTMT YISTGGSIKY EAPLRLGESPHD SEQ NO. 108 SEQ YTDSVKG AFDI QVTLKESGGGLVKPGGSLRLSCAASGFTFSDYTMTW NO. 105 SEQ NO.  SEQ NO. 107 MRQAPGKGLEWVSYISTGGSIKYYTDSVKGRFTISRD 106 NAKNSLYLQMNSLRADDTAVYYCAREAPLRLGESPH DAFDIWGQGTMVTVSS 1.28 DYDMY YISRGGSVT EAPLRLGETPHA SEQ NO. 112 SEQ YYADSVKG AFDI QITLKESGGGLVKPGGSLRLSCAASGFTFSDYDMYWI NO. 109 SEQ SEQ NO. 111 RQAPGKGLEWVSYISRGGSVTYYADSVKGRFTISRD NO. 110 NAKNALYLQMNSLRAEDMAVYFCATEAPLRLGETP HAAFDIWGQGTMVTVSS 1.29 DYTMS YISTGGTIKY EAPLRLGESPHD SEQ NO. 116 SEQ YTDSVKG AFDI QVTLKESGGGLVKPGGSLRLSCAASGFTFSDYTMSW NO. 113 SEQ NO.  SEQ NO. 115 MRQAPGKGLEWVSYISTGGTIKYYTDSVKGRFTISRD 114 NAKNSLYLQMNSLRADDTAVYYCAREAPLRLGESPH DAFDIWGQGTMVTVSS 1.30 DYTMT YISTGGSIKY EAPLRLGESPHD SEQ NO. 120 SEQ YTDSVKG AFDI QVTLKESGGGLVKPGGSLRLSCAASGFTFSDYTMTW NO.  SEQ NO.  SEQ NO. 119 MRQAPGKGLEWVSYISTGGSIKYYTDSVKGRFTISRD 117 118 NARNSLYLQMNSLRAEDTAVYYCAREAPLRLGESPH DAFDIWGQGTMVTVSS 1.31 DYTMT YISTGGSTK EAPLRLGESPHD SEQ NO. 124 SEQ YYTDSVKG AFDI QVTLKESGGGLVKPGGSLRLSCAASGFTFSDYTMTW NO.  SEQ NO.  SEQ NO. 123 MRQAPGKGLEWVSYISTGGSTKYYTDSVKGRFTISR 121 122 DNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGESP HDAFDIWGQGTMVTVSS 1.32 DDYM YISSGGSIIYY EAPLRLGESPHD SEQ NO. 128 M ADSVKG AFDI QVTLKESGGGLVKPGGSLRLSCAASGFTFSDDYMM SEQ SEQ NO.  SEQ NO. 127 WIRQAPGKGLEWVSYISSGGSIIYYADSVKGRFTISR NO.  126 DNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGESP 125 HDAFDIRGQGTMVTVSS 1.33 DYDMY YISRGGSVT EAPLRLGETPHA SEQ NO. 132 SEQ YYADSVKG AFDI QITLKESGGGLVKPGGSLRLSCAASGFTFSDYDMYW NO. 129 SEQ NO.  SEQ NO. 131 VRQAPGKGLEWVSYISRGGSVTYYADSVKGRFTISR 130 DNAKNALYLQMNSLRAEDMAVYFCATEAPLRLGET PHAAFDIWGQGTMVTVSS 1.34 DYTMT YISTGGSVK EAPLRLGESPHD SEQ NO. 136 SEQ YYTDSVKG AFDI EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYTMTW NO.  SEQ NO.  SEQ NO. 135 MRQAPGKGLEWVSYISTGGSVKYYTDSVKGRFTISR 133 134 DNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGESP HDAFDIWGQGTMVTVSS 1.35 DYTMT YISTGGSIKY EAPLRLGESPHD SEQ NO. 140 SEQ YTDSVKG AFDI EVQLLESGGGLVKPGGSLRLSCAASGFTFSDYTMTW NO.  SEQ NO.  SEQ NO. 139 MRQAPGKGLEWVSYISTGGSIKYYTDSVKGRFTISRD 137 138 NAKNSLYLQMNSLRVDDTAVYYCAREAPLRLGESPH DAFDIWGQGTMVTVSS 1.36 DYTMT YISTGGTIKY EAPLRLGESPHD SEQ NO. 144 SEQ YTDSVKG AFDI EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYTMTW NO. 141 SEQ NO.  SEQ NO. 143 MRQAPGKGLEWVSYISTGGTIKYYTDSVKGRFTISRD 142 NAKNSLFLQMNSLRADDTAVYYCAREAPLRLGESPH DAFDIWGQGTMVTVSS 1.37 DYTMT YISTGGSIKY EAPLRLGESPHD SEQ NO. 148 SEQ YTDSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYTMT NO.  SEQ NO.  SEQ NO. 147 WMRQAPGKGLEWVSYISTGGSIKYYTDSVKGRFTIS 145 146 RDNAKNSLFLQMNSLRADDTAVYYCAREAPLRLGES PHDAFDIWGQGTMVTVSS 1.38 DYTMT YISTGGTIKY EAPLRLGESPHD SEQ NO. 152 SEQ YTDSVKG AFDI EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYTMT NO.  SEQ NO.  SEQ NO. 151 WMRQAPGKGLEWVSYISTGGTIKYYTDSVKGRFTIS 149 150 RDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGES PHDAFDIWGQGTMVTVSS 1.39 DYTMT YISTGGSIKY EAPLRLGESPHD SEQ NO. 156 SEQ YTDSVKG AFDI QVQLQESGGGLVKPGGSLRLSCAASGFTFSDYTMT NO.  SEQ SEQ NO. 155 WMRQAPGKGLEWVSYISTGGSIKYYTDSVKGRFTIS 153 NO. 154 RDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGES PHDAFDIWGQGTMVTVSS 1.40 DSSMS YISSGGGIIY EAPLRLGESPHD SEQ NO. 160 SEQ YTDSVKG AFDI EVQLVESGGGLVKPGGSLRLSCAASGFTFSDSSMSW NO.  SEQ NO.  SEQ NO. 159 IRQAPGRGLEWISYISSGGGIIYYTDSVKGRFTISRDN 157 158 AKNSLYLQMNSLRVEDTAVYYCAKEAPLRLGESPHD AFDIWGHGTMVTVSS 1.41 DNSMT YISSGGGVIF EAPLRLGESPHD SEQ NO. 164 SEQ YADSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDNSMT NO.  SEQ NO.  SEQ NO. 163 WMRQAPGKGLEWVSYISSGGGVIFYADSVKGRFTIS 161 162 RDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGES PHDAFDIWGQGTMVTVSS 1.42 DNSMT YISSGGGVK EAPLRLGESPHD SEQ NO. 168 SEQ FYADSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDNSMT NO.  SEQ NO.  SEQ NO. 167 WMRQAPGKGLEWVSYISSGGGVKFYADSVKGRFTI 165 166 SRDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGE SPHDAFDIWGQGTMVTVSS 1.43 DSSMT YISSGGGVIF EAPLRLGESPHD SEQ NO. 172 SEQ YADSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDSSMT NO.  SEQ NO.  SEQ NO. 171 WMRQAPGKGLEWVSYISSGGGVIFYADSVKGRFTIS 169 170 RDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGES PHDAFDIWGQGTMVTVSS 1.44 DNSMT YISSGGAVK EAPLRLGESPHD SEQ NO. 176 SEQ FYADSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDNSMT NO.  SEQ NO.  SEQ NO. 175 WMRQAPGKGLEWVSYISSGGAVKFYADSVKGRFTI 173 174 SRDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGE SPHDAFDIWGQGTMVTVSS 1.45 DYSMS YISSGGGVIF EAPLRLGESPHD SEQ NO. 180 SEQ YADSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYSMS NO.  SEQ NO.  SEQ NO. 179 WMRQAPGKGLEWVSYISSGGGVIFYADSVKGRFTIS 177 178 RDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGES PHDAFDIWGQGTMVTVSS 1.46 DSSMS YISSGGGVIF EAPLRLGESPHD SEQ NO. 184 SEQ YADSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDSSMS NO.  SEQ NO.  SEQ NO. 183 WMRQAPGKGLEWVSYISSGGGVIFYADSVKGRFTIS 181 182 RDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGES PHDAFDIWGQGTMVTVSS 1.47 DNSMS YISSGGGVIF EAPLRLGESPHD SEQ NO. 188 SEQ YADSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDNSMS NO.  SEQ NO.  SEQ NO. 187 WMRQAPGKGLEWVSYISSGGGVIFYADSVKGRFTIS 185 186 RDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGES PHDAFDIWGQGTMVTVSS 1.48 DSSMT YISSGGGVK EAPLRLGESPHD SEQ NO. 192 SEQ FYADSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDSSMT NO.  SEQ NO.  SEQ NO. 191 WMRQAPGKGLEWVSYISSGGGVKFYADSVKGRFTI 189 190 SRDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGE SPHDAFDIWGQGTMVTVSS 1.49 DSSMS YISTGGGVK EAPLRLGESPHD SEQ NO. 196 SEQ FYADSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDSSMS NO.  SEQ NO.  SEQ NO. 195 WMRQAPGKGLEWVSYISTGGGVKFYADSVKGRFTI 193 194 SRDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGE SPHDAFDIWGQGTMVTVSS 1.50 DNSMT YISSGGTIKF EAPLRLGESPHD SEQ NO. 200 SEQ YADSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDNSMT NO.  SEQ NO.  SEQ NO. 199 WMRQAPGKGLEWVSYISSGGTIKFYADSVKGRFTIS 197 198 RDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGES PHDAFDIWGQGTMVTVSS 1.51 DSSMT YISSGGAVK EAPLRLGESPHD SEQ NO. 204 SEQ FYTDSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDSSMT NO.  SEQ NO.  SEQ NO. 203 WMRQAPGKGLEWVSYISSGGAVKFYTDSVKGRFTI 201 202 SRDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGE SPHDAFDIWGQGTMVTVSS 1.52 5 DNSMT YISSGGGVK EAPLRLGESPHD SEQ NO. 208 SEQ YYADSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDNSMT NO.  SEQ NO.  SEQ NO. 207 WMRQAPGKGLEWVSYISSGGGVKYYADSVKGRFTI 205 206 SRDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGE SPHDAFDIWGQGTMVTVSS 1.53 DNSMT YISSGGSVK EAPLRLGESPHD SEQ NO. 212 SEQ FYADSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDNSMT NO. 209 SEQ NO.  SEQ NO. 211 WMRQAPGKGLEWVSYISSGGSVKFYADSVKGRFTI 210 SRDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGE SPHDAFDIWGQGTMVTVSS 1.54 DDSMT YISSGGGVIF EAPLRLGESPHD SEQ NO. 216 SEQ YADSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDDSMT NO.  SEQ NO.  SEQ NO. 215 WMRQAPGKGLEWVSYISSGGGVIFYADSVKGRFTIS 213 214 RDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGES PHDAFDIWGQGTMVTVSS 1.55 DNSMT YISSGGGVK EAPLRLGESPHD SEQ NO. 220 SEQ FYADSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDNSMT NO.  SEQ NO.  SEQ NO. 219 WMRQAPGKGLEWVSYISSGGGVKFYADSVKGRFTI 217 218 SRDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGE SPHDAFDI SGQGTMVTVSS 1.56 DNSMT YISSGGAVK EAPLRLGESPHD SEQ NO. 224 SEQ FYADSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDNSMT NO.  SEQ NO.  SEQ NO. 223 WMRQAPGKGLEWVSYISSGGAVKFYADSVKGRFTI 221 222 SRDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGE SPHDAFDILGQGTMVTVSS 1.57 DNSMT YISSGGGVIF EAPLRLGESPHD SEQ NO. 228 SEQ YADSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDNSMT NO.  SEQ NO.  SEQ NO. 227 WMRQAPGKGLEWVSYISSGGGVIFYADSVKGRFTIS 225 226 RDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGES PHDAFDISGQGTMVTVSS 1.58 DNTMT YISTGGGVK EAPLRLGESPHD SEQ NO. 232 SEQ FYADSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYTMT NO. 229 SEQ NO.  SEQ NO. 231 WMRQAPGKGLEWVSYISTGGTIKYYTDSVKGRFTIS 230 RDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGES PHDAFDIWGQGTMVTVSS 1.59 DNSMS YISSGGSVK EAPLRLGESPHD SEQ NO. 236 SEQ FYADSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDNTMT NO.  SEQ NO.  SEQ NO. 235 WMRQAPGKGLEWVSYISTGGGVKFYADSVKGRFTI 233 234 SRDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGE SPHDAFDIWGQGTMVTVSS 1.60 DNSMT YISTGGGVK EAPLRLGESPHD SEQ NO. 240 SEQ YYADSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDNSMS NO.  SEQ NO.  SEQ NO. 239 WMRQAPGKGLEWVSYISSGGSVKFYADSVKGRFTI 237 238 SRDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGE SPHDAFDIWGQGTMVTVSS 1.61 DYTMS YISTGGGVK EAPLRLGESPHD SEQ NO. 244 SEQ FYADSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDNSMT NO.  SEQ NO.  SEQ NO. 243 WMRQAPGKGLEWVSYISTGGGVKYYADSVKGRFTI 241 242 SRDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGE SPHDAFDIWGQGTMVTVSS 1.62 DSSMT YISSGGAVK EAPLRLGESPHD SEQ NO. 248 SEQ FYTDSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDSSMT NO.  SEQ NO.  SEQ NO. 247 WMRQAPGKGLEWVSYISSGGAVKFYTDSVKGRFTI 245 246 SRDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGE SPHDAFDISGQGTMVTVSS 1.63 DSSMT YISSGGGVK EAPLRLGESPHD SEQ NO. 252 SEQ FYTDSVKG AFDI EVQLVESGGGLVKPGGSLRLSCAASGFTFSDSSMTW NO.  SEQ NO.  SEQ NO. 251 MRQAPGKGLEWVSYISSGGGVKFYTDSVKGRFTISR 249 250 DNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGESP HDAFDISGQGTMVTVSS 1.64 DSSMT YISSGGGVK EAPLRLGESPHD SEQ NO. 256 SEQ FYADSVKG AFDI EVQLVESGGGLVKPGGSLRLSCAASGFTFSDSSMTW NO.  SEQ NO.  SEQ NO. 255 MRQAPGKGLEWVSYISSGGGVKFYADSVKGRFTISR 253 254 DNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGESP HDAFDISGQGTMVTVSS 1.65 DSSMT YISSGGGVK EAPLRLGESPHD SEQ NO. 260 SEQ FYADSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDSSMT NO.  SEQ NO.  SEQ NO. 259 WMRQAPGKGLEWVSYISSGGGVKFYADSVKGRFTI 257 258 SRDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGE SPHDAFDISGQGTMVTVSS 1.66 DSSMT YISSGGGVK EAPLRLGESPHD SEQ NO. 264 SEQ FYTDSVKG AFDT QVQLVESGGGLVKPGGSLRLSCAASGFTFSDSSMT NO.  SEQ NO.  SEQ NO. 263 WMRQAPGKGLEWVSYISSGGGVKFYTDSVKGRFTI 261 262 SRDNAKNSLYLQMDSLRADDTAVYYCAREAPLRLGE SPHDAFDTSGQGTMVTVSS 1.67 GSSMT YISSGGGVIF EAPLRLGESPHD SEQ NO. 268 SEQ YADSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSGSSMT NO.  SEQ NO.  SEQ NO. 267 WMRQAPGKGLEWVSYISSGGGVIFYADSVKGRFTIS 265 266 RDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGES PHDAFDISGQGTMVTVSS 1.68 DNSMT YISSGGGVIF EAPLRLGESPHD SEQ NO. 272 SEQ YADSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFGDNSMT NO.  SEQ NO.  SEQ NO. 271 WMRQAPGKGLEWVSYISSGGGVIFYADSVKGRFTIS 269 270 RDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGES PHDAFDISGRGTTVTVSS 1.69 DNSMS YISSGGGVIF EAPLRLGESPHD SEQ NO. 276 SEQ YADSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDNSMS NO.  SEQ NO.  SEQ NO. 275 WMRQAPGKGLEWVSYISSGGGVIFYADSVKGRFTIS 273 274 RDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGES PHDAFDISGQGTMVTVSS 1.70 DSSMT YISSGGAVK EAPLRLGESPHD SEQ NO. 280 SEQ FYTDSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFGDSSMT NO.  SEQ NO.  SEQ NO. 279 WMRQAPGKGLEWVSYISSGGAVKFYTDSVKGRFTI 277 278 SRDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGE SPHDAFDISGQGTMVTVSS 1.71 GSSMT YISSGGGVK EAPLRLGESPHD SEQ NO. 284 SEQ FYTDSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFGGSSMT NO.  SEQ NO.  SEQ NO. 283 WMRQAPGKGLEWVSYISSGGGVKFYTDSVKGRFTI 281 282 SRDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGE SPHDAFDISGQGTMVTVSS 1.72 DSSMS YISSGGGVIF EAPLRLGESPHD SEQ NO. 288 SEQ YADSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAASGFTFSDSSMS NO.  SEQ NO.  SEQ NO. 287 WMRQAPGKGLEWVSYISSGGGVIFYADSVKGRFTIS 285 286 RDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGES PHDAFDISGQGTMVTVSS 1.73 DSSMT YISAGGGVR EAPLRLGESPHD SEQ NO. 292 SEQ FYTDSVKG AFDI QVQLVESGGGLVKPGGSLRLSCAATGFTFSDSSMT NO.  SEQ NO.  SEQ NO. 291 WMRQAPGKGLEWVSYISAGGGVRFYTDSVKGRFTI 289 290 SRDNAKNSLYLQMNSLRADDTAVYYCAREAPLRLGE SPHDAFDISGQGTMVTVSS 1.74 SEQ ID SEQ ID NO: SEQ ID NO: 368 SEQ ID NO: 369 NO: 367 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDSSMTW 366 YISSGGGVK AFDI IRQAPGKGLEWVSYISSGGGVKFYTDSVKGRFTISRD DSSMT FYTDSVKG NAKNSLYLQMNSLRADDTAVYYCAREAPLRLGESPH DAFDISGQGTMVTVSS 1.75 SEQ ID SEQ ID NO: SEQ ID NO: 372 SEQ ID NO: 373 NO: 371 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDSSMTW 370 YISSGGGVK AFDI IRQAPGKGLEWVSYISSGGGVKFYTDSVKGRFTISRD DSSMT FYTDSVKG NAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGESPH DAFDISGQGTMVTVSS 1.76 SEQ ID SEQ ID NO: SEQ ID NO: 376 SEQ ID NO: 377 NO: 375 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDSSMTW 374 YISSGGGVK AFDI IRQAPGKGLEWVSYISSGGGVKFYADSVKGRFTISRD DSSMT FYADSVKG NAKNSLYLQMNSLRADDTAVYYCAREAPLRLGESPH DAFDISGQGTMVTVSS 1.77 SEQ ID SEQ ID NO: SEQ ID NO: 380 SEQ ID NO: 381 NO: 379 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDSSMTW 378 YISSGGGVK AFDI MRQAPGKGLEWVSYISSGGGVKFYTDSVKGRFTISR DSSMT FYTDSVKG DNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGESP HDAFDISGQGTMVTVSS 1.78 SEQ ID SEQ ID NO: SEQ ID NO: 384 SEQ ID NO: 385 NO: 383 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDSSMTW 382 YISSGGGVK AFDI IRQAPGKGLEWVSYISSGGGVKFYADSVKGRFTISRD DSSMT FYADSVKG NAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGESPH DAFDISGQGTMVTVSS 1.79 SEQ ID SEQ ID NO: SEQ ID NO: 388 SEQ ID NO: 389 NO: 387 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDSSMTW 386 YISSGGGVK AFDI KRQAPGKGLEWVSYISSGGGVKFYADSVKGRFTISR DSSMT FYADSVKG DNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGESP HDAFDISGQGTMVTVSS 1.80 SEQ ID SEQ ID NO: SEQ ID NO: 392 SEQ ID NO: 393 NO: 391 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDSSMTW 390 YISSGGGVK AFDI MRQAPGKGLEWVSYISSGGGVKFYADSVKGRFTISR DSSMT FYADSVKG DNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGESP HDAFDISGQGTMVTVSS 1.81 SEQ ID SEQ ID NO: SEQ ID NO: 396 SEQ ID NO: 397 NO: 395 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDSSMTW 394 YISSGGGVK AFDI VRQAPGKGLEWVSYISSGGGVKFYADSVKGRFTISR DSSMT FYADSVKG DNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGESP HDAFDISGQGTMVTVSS 1.82 SEQ ID SEQ ID NO: SEQ ID NO: 400 SEQ ID NO: 401 NO: 399 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDTSMT 398 YISSGGGVK AFDI WMRQAPGKGLEWVSYISSGGGVKFYTDSVKGRFTI DTSMT FYTDSVKG SRDNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGE SPHDAFDISGQGTMVTVSS 1.83 SEQ ID SEQ ID NO: SEQ ID NO: 404 SEQ ID NO: 405 NO: 403 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDESMT 402 YISSGGGVK AFDI WFRQAPGKGLEWVSYISSGGGVKFYTDSVKGRFTIS DESMT FYTDSVKG RDNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGES PHDAFDISGQGTMVTVSS 1.84 SEQ ID SEQ ID NO: SEQ ID NO: 408 SEQ ID NO: 409 NO: 407 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDESMT 406 YISSGGGVK AFDI WMRQAPGKGLEWVSYISSGGGVKFYTDSVKGRFTI DESMT FYTDSVKG SRDNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGE SPHDAFDISGQGTMVTVSS 1.85 SEQ ID SEQ ID NO: SEQ ID NO: 412 SEQ ID NO: 413 NO: 411 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYSMT 410 YISSGGGVK AFDI WMRQAPGKGLEWVSYISSGGGVKFYTDSVKGRFTI DYSMT FYTDSVKG SRDNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGE SPHDAFDISGQGTMVTVSS 1.86 SEQ ID SEQ ID NO: SEQ ID NO: 416 SEQ ID NO: 417 NO: 415 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDASMT 414 YISSGGGVK AFDI WMRQAPGKGLEWVSYISSGGGVKFYTDSVKGRFTI DASMT FYTDSVKG SRDNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGE SPHDAFDISGQGTMVTVSS 1.87 SEQ ID SEQ ID NO: SEQ ID NO: 420 SEQ ID NO: 421 NO: 419 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDKSMT 418 YISSGGGVK AFDI WMRQAPGKGLEWVSYISSGGGVKFYTDSVKGRFTI DKSMT FYTDSVKG SRDNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGE SPHDAFDISGQGTMVTVSS 1.88 SEQ ID SEQ ID NO: SEQ ID NO: 424 SEQ ID NO: 425 NO: 423 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDRSMT 422 YISSGGGVK AFDI WMRQAPGKGLEWVSYISSGGGVKFYTDSVKGRFTI DRSMT FYTDSVKG SRDNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGE SPHDAFDISGQGTMVTVSS 1.89 SEQ ID SEQ ID NO: SEQ ID NO: 428 SEQ ID NO: 429 NO: 427 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYSMT 426 YISSGGGVK AFDI WMRQAPGKGLEWVSYISSGGGVKFYADSVKGRFTI DYSMT FYADSVKG SRDNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGE SPHDAFDISGQGTMVTVSS 1.90 SEQ ID SEQ ID NO: SEQ ID NO: 432 SEQ ID NO: 433 NO: 431 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDVSMT 430 YISSGGGVK AFDI WMRQAPGKGLEWVSYISSGGGVKFYADSVKGRFTI DVSMT FYADSVKG SRDNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGE SPHDAFDISGQGTMVTVSS 1.91 SEQ ID SEQ ID NO: SEQ ID NO: 436 SEQ ID NO: 437 NO: 435 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDQSMT 434 YISSGGGVK AFDI WMRQAPGKGLEWVSYISSGGGVKFYADSVKGRFTI DQSMT FYADSVKG SRDNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGE SPHDAFDISGQGTMVTVSS 1.92 SEQ ID SEQ ID NO: SEQ ID NO: 440 SEQ ID NO: 441 NO: 439 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDESMT 438 YISSGGGVK AFDI WMRQAPGKGLEWVSYISSGGGVKFYADSVKGRFTI DESMT FYADSVKG SRDNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGE SPHDAFDISGQGTMVTVSS 1.93 SEQ ID SEQ ID NO: SEQ ID NO: 444 SEQ ID NO: 445 NO: 443 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDASMT 442 YISSGGGVK AFDI WMRQAPGKGLEWVSYISSGGGVKFYADSVKGRFTI DASMT FYADSVKG SRDNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGE SPHDAFDISGQGTMVTVSS 1.94 SEQ ID SEQ ID NO: SEQ ID NO: 448 SEQ ID NO: 449 NO: 447 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDWSMT 446 YISSGGGVK AFDI WMRQAPGKGLEWVSYISSGGGVKFYADSVKGRFTI DWSM FYADSVKG SRDNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGE T SPHDAFDISGQGTMVTVSS 1.95 SEQ ID SEQ ID NO: SEQ ID NO: 452 SEQ ID NO: 453 NO: 451 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDGSMT 450 YISSGGGVK AFDI WMRQAPGKGLEWVSYISSGGGVKFYADSVKGRFTI DGSMT FYADSVKG SRDNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGE SPHDAFDISGQGTMVTVSS 1.96 SEQ ID SEQ ID NO: SEQ ID NO: 456 SEQ ID NO: 457 NO: 455 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDTSMT 454 YISSGGGVK AFDI WMRQAPGKGLEWVSYISSGGGVKFYADSVKGRFTI DTSMT FYADSVKG SRDNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGE SPHDAFDISGQGTMVTVSS 1.97 SEQ ID SEQ ID NO: SEQ ID NO: 460 SEQ ID NO: 461 NO: 459 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDISMTW 458 YISSGGGVK AFDI MRQAPGKGLEWVSYISSGGGVKFYADSVKGRFTISR DISMT FYADSVKG DNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGESP HDAFDISGQGTMVTVSS 1.98 SEQ ID SEQ ID NO: SEQ ID NO: 464 SEQ ID NO: 465 NO: 463 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDKSMT 462 YISSGGGVK AFDI WMRQAPGKGLEWVSYISSGGGVKFYADSVKGRFTI DKSMT FYADSVKG SRDNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGE SPHDAFDISGQGTMVTVSS 1.99 SEQ ID SEQ ID NO: SEQ ID NO: 468 SEQ ID NO: 469 NO: 467 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDRSMT 466 YISSGGGVK AFDI WMRQAPGKGLEWVSYISSGGGVKFYADSVKGRFTI DRSMT FYADSVKG SRDNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGE SPHDAFDISGQGTMVTVSS 1.100 SEQ ID SEQ ID NO: SEQ ID NO: 472 SEQ ID NO: 473 NO: 471 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDLSMTW 470 YISSGGGVK AFDI MRQAPGKGLEWVSYISSGGGVKFYADSVKGRFTISR DLSMT FYADSVKG DNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGESP HDAFDISGQGTMVTVSS 1.101 SEQ ID SEQ ID NO: SEQ ID NO: 476 SEQ ID NO: 477 NO: 475 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDFSMTW 474 YISSGGGVK AFDI MRQAPGKGLEWVSYISSGGGVKFYADSVKGRFTISR DFSMT FYADSVKG DNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGESP HDAFDISGQGTMVTVSS 1.102 SEQ ID SEQ ID NO: SEQ ID NO: 480 SEQ ID NO: 481 NO: 479 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDESVTW 478 YISSGGGVK AFDI MRQAPGKGLEWVSYISSGGGVKFYTDSVKGRFTISR DESVT FYTDSVKG DNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGESP HDAFDISGQGTMVTVSS 1.103 SEQ ID SEQ ID NO: SEQ ID NO: 484 SEQ ID NO: 485 NO: 483 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDESQTW 482 YISSGGGVK AFDI MRQAPGKGLEWVSYISSGGGVKFYTDSVKGRFTISR DESQT FYTDSVKG DNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGESP HDAFDISGQGTMVTVSS 1.104 SEQ ID SEQ ID NO: SEQ ID NO: 488 SEQ ID NO: 489 NO: 487 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDESFTW 486 YISSGGGVK AFDI MRQAPGKGLEWVSYISSGGGVKFYTDSVKGRFTISR DESFT FYTDSVKG DNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGESP HDAFDISGQGTMVTVSS 1.105 SEQ ID SEQ ID NO: SEQ ID NO: 492 SEQ ID NO: 493 NO: 491 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDESLTW 490 YISSGGGVK AFDI MRQAPGKGLEWVSYISSGGGVKFYTDSVKGRFTISR DESLT FYTDSVKG DNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGESP HDAFDISGQGTMVTVSS 1.106 SEQ ID SEQ ID NO: SEQ ID NO: 496 SEQ ID NO: 497 NO: 495 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDESKTW 494 YISSGGGVK AFDI MRQAPGKGLEWVSYISSGGGVKFYTDSVKGRFTISR DESKT FYTDSVKG DNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGESP HDAFDISGQGTMVTVSS 1.107 SEQ ID SEQ ID NO: SEQ ID NO: 500 SEQ ID NO: 501 NO: 499 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDESYTW 498 YISSGGGVK AFDI MRQAPGKGLEWVSYISSGGGVKFYTDSVKGRFTISR DESYT FYTDSVKG DNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGESP HDAFDISGQGTMVTVSS 1.108 SEQ ID SEQ ID NO: SEQ ID NO: 504 SEQ ID NO: 505 NO: 503 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDESATW 502 YISSGGGVK AFDI MRQAPGKGLEWVSYISSGGGVKFYADSVKGRFTISR DESAT FYADSVKG DNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGESP HDAFDISGQGTMVTVSS 1.109 SEQ ID SEQ ID NO: SEQ ID NO: 508 SEQ ID NO: 509 NO: 507 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDESFTW 506 YISSGGGVK AFDI MRQAPGKGLEWVSYISSGGGVKFYADSVKGRFTISR DESFT FYADSVKG DNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGESP HDAFDISGQGTMVTVSS 1.110 SEQ ID SEQ ID NO: SEQ ID NO: 512 SEQ ID NO: 513 NO: 511 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDESNTW 510 YISSGGGVK AFDI MRQAPGKGLEWVSYISSGGGVKFYADSVKGRFTISR DESNT FYADSVKG DNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGESP HDAFDISGQGTMVTVSS 1.111 SEQ ID SEQ ID NO: SEQ ID NO: 516 SEQ ID NO: 517 NO: 515 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDESWT 514 YISSGGGVK AFDI WMRQAPGKGLEWVSYISSGGGVKFYADSVKGRFTI DESWT FYADSVKG SRDNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGE SPHDAFDISGQGTMVTVSS 1.112 SEQ ID SEQ ID NO: SEQ ID NO: 520 SEQ ID NO: 521 NO: 519 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDESITW 518 YISSGGGVK AFDI MRQAPGKGLEWVSYISSGGGVKFYADSVKGRFTISR DESIT FYADSVKG DNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGESP HDAFDISGQGTMVTVSS 1.113 SEQ ID SEQ ID NO: SEQ ID NO: 524 SEQ ID NO: 525 NO: 523 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDESSTW 522 YISSGGGVK AFDI MRQAPGKGLEWVSYISSGGGVKFYADSVKGRFTISR DESST FYADSVKG DNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGESP HDAFDISGQGTMVTVSS 1.114 SEQ ID SEQ ID NO: SEQ ID NO: 528 SEQ ID NO: 529 NO: 527 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDESHTW 526 YISSGGGVK AFDI MRQAPGKGLEWVSYISSGGGVKFYADSVKGRFTISR DESHT FYADSVKG DNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGESP HDAFDISGQGTMVTVSS 1.115 SEQ ID SEQ ID NO: SEQ ID NO: 532 SEQ ID NO: 533 NO: 531 EAPLRLGESPHD EVQLVESGGGLVKPGGSLRLSCAASGFTFSDESGTW 530 YISSGGGVK AFDI MRQAPGKGLEWVSYISSGGGVKFYADSVKGRFTISR DESGT FYADSVKG DNAKNSLYLQMNSLRAEDTAVYYCAREAPLRLGESP HDAFDISGQGTMVTVSS

In some embodiments, the invention provides a V_(H) single domain antibody that is a variant of any of the above single V_(H) domain antibodies having one or more amino acid substitutions, deletions, insertions or other modifications, and which retains a biological function of the single domain antibody. Thus, variant V_(H) single domain antibody can be sequence engineered. Modifications may include one or more substitution, deletion or insertion of one or more codons encoding the single domain antibody or polypeptide that results in a change in the amino acid sequence as compared with the native sequence V_(H) single domain antibody or polypeptide. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence. A variant of a V_(H) single domain antibody described herein has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to the non-variant molecule, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology.

In one embodiment, the modification is a conservative sequence modification. As used herein, the term “conservative sequence modifications” is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of a single domain antibody of the invention can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for retained function (i.e., the functions set forth in (c) through (I) above) using the functional assays described herein.

In some embodiments, the invention provides a V_(H) single domain antibody that is a variant of a single domain antibody selected from those shown in Table 1 that comprises one or more sequence modification and has improvements in one or more of a property such as binding affinity, specificity, thermostability, expression level, effector function, glycosylation, reduced immunogenicity, or solubility as compared to the unmodified single domain antibody.

A skilled person will know that there are different ways to identify, obtain and optimise the antigen binding molecules as described herein, including in vitro and in vivo expression libraries. This is further described in the examples. Optimisation techniques known in the art, such as display (e.g., ribosome and/or phage display) and/or mutagenesis (e.g., error-prone mutagenesis) can be used. The invention therefore also comprises sequence optimised variants of the single domain antibodies described herein.

In one embodiment, modifications can be made to decrease the immunogenicity of the single domain antibody. For example, one approach is to revert one or more framework residues to the corresponding human germline sequence. More specifically, a single domain antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the single domain antibody is derived. Such residues can be identified by comparing the single domain antibody framework sequences to the germline sequences from which the single domain antibody is derived.

To return one or more of the amino acid residues in the framework region sequences to their germline configuration, the somatic mutations can be “backmutated” to the germline sequence by, for example, site-directed mutagenesis or PCR-mediated mutagenesis.

Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody.

In still another embodiment, the glycosylation of an antibody is modified. For example, an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen.

In one embodiment, the variant V_(H) single domain antibody is selected from any one of SEQ ID Nos. 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 272, 276, 280, 284, 288 or 292, but comprises one or more amino acid substitutions, for example 1 to 20 for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions compared to these sequences. In one embodiment, the variant V_(H) single domain antibody is selected from SEQ ID No. 4. In one embodiment, the variant V_(H) single domain antibody is selected from SEQ ID No. 228, 256 or 441. In one embodiment, the one or more amino acid substitution is in one or more of the framework areas. In another embodiment, the one or more amino acid substitution is in one or more of the CDRs. In one embodiment, the amino acid substitutions are in the framework and CDR sequences. In one embodiment, the single domain antibody comprises or consists of SEQ ID No. 4, 228, 441 or a sequence which comprises one or more amino acid substitutions, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions.

Thus, these amino acid changes can typically be made without altering the biological activity, function, or other desired property of the polypeptide, such as its affinity or its specificity for antigen. In general, single amino acid substitutions in nonessential regions of a polypeptide do not substantially alter biological activity. Furthermore, substitutions of amino acids that are similar in structure or function are less likely to disrupt the polypeptides' biological activity. Abbreviations for the amino acid residues that comprise polypeptides and peptides described herein, and conservative substitutions for these amino acid residues are shown in Table 3 below.

TABLE 3 Amino Acid Residues and Examples of Conservative Amino Acid Substitutions Original residue Three letter code, single letter code Conservative substitution Alanine, Ala, A Gly, Ser Arginine, Arg, R Lys, His Asparagine, Asn, N Gln, His Aspartic acid Asp, D Glu, Asn Cysteine, Cys, C Ser, Ala Glutamine, Gln, Q Asn Glutamic acid, Glu, E Asp, Gln Glycine, Gly, G Ala Histidein, His, H Asn, Gln Isoleucine, Ile, I Leu, Val Leucine, Leu, L Ile, Val Lysine, lys, K Ar, His Methionine, Met, M Leu, Ile, Tyr Phenylalanine, Phe, F Tyr, Met, Leu Proline, Pro, P Ala Serine, Ser, S Thr Threonine, Thr, T Se Tryptophan, Trp, W Tyr, Phe Tyrosine, Tyr, Y Try, Phe Valine, Val, V Ile, Leu

In one embodiment, a Q at position 1 is replaced with E.

In one embodiment, the variant V_(H) single domain antibody comprises SEQ ID No. 4 (Humabody® 1.1) but with amino acid substitutions at one or more the following positions: Y32, T33, T53, T56, I57, K58, Y59, T61 and/or W115.

In one embodiment, the V_(H) single domain antibody comprises SEQ ID No. 4 (Humabody® 1.1) but with the following amino acid substitutions: Y32→N, T33→S, T53→S, T56→G, 157→N, K58→I, Y59→F, W115→S (Humabody® 1.57)

In one embodiment, the variant V_(H) single domain antibody comprises SEQ ID No. 228 (Humabody® 1.57) but with amino acid substitutions at one or more or all of the following positions: D31, N32, I58, A61, T35, S30, G56, S25Q117, M120 and/or Q1.

In one embodiment, the variant V_(H) single domain antibody comprises SEQ ID No. 228 (Humabody® 1.57) but with amino acid substitutions selected from one of the following

-   -   1) D31→G, N32→S (Humabody® 1.67);     -   2) S30→G, D31→G, N32→S, I58→K, A61→T (Humabody® 1.71);     -   3) T35→S (1.69);     -   4) S30→G, N32→S, I58→K, A61→T (Humabody® 1.70);     -   5) S25→J, N32→S, I58→R, A61→T (Humabody® 1.73);     -   6) N32→S, I58→X, N84→D, I114→J (Humabody® 1.66);     -   7) S30→G, Q117→R, M120→J (Humabody® 1.68);     -   8) N32→S, T35→S (Humabody® 1.72);     -   9) N32→S, G56→A, I58→K, A61→T (Humabody® 1.62);     -   10) N32→S, I58→K (Humabody® 1.65);     -   11) Q1→E, N32→S, I58→K (Humabody® 1.64);     -   12) Q1→E, N32→S, I58→K, A61→T (Humabody® 1.63).

In one embodiment, the variant V_(H) single domain antibody comprises SEQ ID No. 256 (Humabody® 1.64) but with amino acid substitutions at one or more or all of the following positions: S32, D90 and/or A61.

In one embodiment, the variant V_(H) single domain antibody comprises SEQ ID No. 256 (Humabody® 1.64) but with amino acid substitutions selected from one of the following

-   -   1. S32→E, D90→E (Humabody® 1.92)     -   2. S32→R, D90→E (Humabody® 1.99)     -   3. S32→N, D90→E (Humabody® 1.89)     -   4. S32→E, D90→E A61→T (Humabody® 1.84)     -   5. D85→E A60→T (Humabody® 1.77)     -   6. S32→T, D90→E A60→T (Humabody® 1.82)

The numbering used above is based on the actual position of the residue.

V_(H) single domain antibodies of the invention have shown excellent stability. Furthermore, V_(H) single domain antibodies of the invention also show specificity for human PD-1, bind to cyno PD-1 and have fast on rates (see examples).

The V_(H) single domain antibodies of the invention preferably have KD, IC₅₀ and/or EC₅₀ values as further described herein and as shown in the examples. V_(H) single domain antibodies specifically bind to human PD-1 with high affinity, the KD can be least about 10⁻⁸ M to 10⁻¹² such as 10⁻⁸M, 10⁻⁹M, 10⁻¹⁰ M, 10⁻¹¹ M or 10⁻¹²M as measured by surface plasmon resonance (biomolecular interaction analysis, BIAcore®) analysis. Single domain antibodies according to the invention preferably has an IC50 value for inhibition of PD-1 in 10⁻⁸ M to 10⁻⁹ M range as determined in functional assay.

The term “KD” refers to the “equilibrium dissociation constant” and refers to the value obtained in a titration measurement at equilibrium, or by dividing the dissociation rate constant (Koff) by the association rate constant (Kon). “KA” refers to the affinity constant. The association rate constant, the dissociation rate constant and the equilibrium dissociation constant are used to represent the binding affinity of an antibody to an antigen. Methods for determining association and dissociation rate constants are well known in the art. Using fluorescence-based techniques offers high sensitivity and the ability to examine samples in physiological buffers at equilibrium. Other experimental approaches and instruments such as a BIAcore® assay can be used.

The present invention further provides an isolated nucleic acid encoding a single domain antibody of the present invention. Nucleic acid may include DNA and/or RNA. In one aspect, the present invention provides a nucleic acid that codes for a CDR, for example CDR3, a set of two or three CDRs or for a V_(H) single domain antibody as shown in Table 1 and as further defined above.

In one aspect, the invention thus also relates to a nucleic acid sequences comprising or consisting of a sequence selected from SEQ ID Nos. 293 to 365 and 534 to 575. These nucleic acid sequences encode V_(H) single domain antibodies as shown in Table 1.

In one embodiment, the nucleic acid sequence has at least 60%, 70%, 80%, 90%, 95% or more sequence homology to a sequence selected from SEQ ID Nos. 293 to 365. In one embodiment, said sequence homology is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

A nucleic acid according to the present invention may comprise DNA or RNA and may be wholly or partially synthetic or recombinantly produced. Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence, and encompasses a RNA molecule with the specified sequence in which U is substituted for T, unless context requires otherwise.

Furthermore, the invention relates to a nucleic acid construct comprising at least one nucleic acid as defined above. The construct may be in the form of a plasmid, vector, transcription or expression cassette.

The invention also relates to an isolated recombinant host cell comprising one or more nucleic acid construct as described above. The host cell may be a bacterial, viral, insect, plant, mammalian or other suitable host cell. In one embodiment, the cell is an E. coli cell. In another embodiment, the cell is a yeast cell. In another embodiment, the cell is a Chinese Hamster Ovary (CHO) cell.

In one embodiment, a method of making an anti-PD-1 single domain antibody as described herein is provided, wherein the method comprises culturing the host cell under conditions suitable for expression of the polynucleotide encoding the single domain antibody, and isolating the single domain antibody.

In another aspect, the invention provides binding molecules, e.g. antibodies, antibody fragments or antibody mimetics that bind to the same epitope on human PD-1 as any of the PD-1 single domain antibodies of the invention (i.e. antibodies that have the ability to cross-compete for binding to PD-1 with any of the single domain antibodies of Table 1. The single domain antibodies of the invention can thus be used as a reference antibody). In preferred embodiments, the reference antibody for cross-competition studies is single domain antibody 1.1 (SEQ ID No. 4), 1.57 (SEQ ID No. 228) or 1.92 (SEQ ID NO. 441).

Such cross-competing antibodies can be identified based on their ability to cross-compete with any of single domain antibodies 1.1 to 1.115 in standard PD-1 binding assays. For example, BIAcore® analysis, ELISA assays or flow cytometry may be used to demonstrate cross-competition with the single domain antibodies of the current invention.

In one embodiment, the invention provides a binding agent capable of binding human PD-1 wherein any one of the single domain antibodies described above displaces the binding agent in a competitive assay. In one embodiment, said single domain antibody is selected from 1.1 (SEQ ID No. 4), 1.57 (SEQ ID No. 228) or 1.92 (SEQ ID NO. 441). In some embodiments, the binding agent is an antibody, a functional fragment thereof, for example a single domain antibody, or an antibody mimetic protein. In another aspect, invention provides a binding agent capable of binding human PD-1 wherein the binding agent displaces any one of the single domain antibodies described above in a competitive assay. In one embodiment, said single domain antibody is selected from SEQ ID No. 1.1 (SEQ ID No. 4), 1.57 (SEQ ID No. 228), or 1.92 (SEQ ID NO. 441). In another aspect, invention provides a binding agent capable of binding human PD-1 wherein the binding agent binds to essentially the same epitope as the single domain antibody of the invention.

In another aspect, the invention provides an isolated heavy chain only antibody comprising a V_(H) domain as described herein and listed in Table 1 or with at least 70%, 80% or 90% homology thereto.

In one aspect, the invention relates to a binding agent comprising a single domain antibody according to the invention and at least a second moiety. Thus, the invention also provides multifunctional molecules. In one embodiment, the at least second moiety is a binding molecule, for example selected from an antibody or antibody fragment (e.g., a Fab, F(ab′)2, Fv, a single chain Fv fragment (scFv) or single domain antibody, for example a V_(H) domain) or antibody mimetic protein. In one embodiment, the at least second moiety is a V_(H) domain. In one embodiment, the single domain antibody of the invention can be linked to an antibody Fc region or fragment thereof, comprising one or both of C_(H)2 and C_(H)3 domains, and optionally a hinge region.

The binding agent may be multivalent, for example bivalent, or multiparatopic, for example biparatopic. Thus, the binding molecule may comprise a first V_(H) single domain antibody and V_(H)(A) and a second V_(H) single domain antibody and V_(H)(B) and thus has the following formula: V_(H)(A)-V_(H)(B).

Each V_(H) comprises CDR and FR regions. Thus, the binding molecule may have the following formula: FR1(A)-CDR1(A)-FR2(A)-CDR2(A)-FR3(A)-CDR3(A)-FR4(A)-FR1(B)-CDR1(B)-FR2(B)-CDR2(BA)-FR3(B)-CDR3(B)-FR4(B). The order of the immunoglobulin single variable domains A and B is not particularly limited, so that, within a polypeptide of the invention, immunoglobulin single variable domain A may be located N-terminally and immunoglobulin single variable domain B may be located C-terminally, or vice versa. The V_(H) domain antibodies are typically connected via a linker.

In one embodiment, the binding molecule is biparatopic or bispecific. Thus, in one aspect, the invention relates to a bispecific molecule comprising the single domain antibody described herein linked to a second functional moiety having a different binding specificity than said single domain antibody.

In one embodiment, biparatopic binding molecules are provided that comprise a first and a second binding molecule that bind to the target protein PD-1, but on different or overlapping sites. Complete or partial blocking can be seen in epitope binning studies. The first binding molecule is a single domain antibody according to the invention. In one embodiment, the second binding molecule is a PD-1 inhibitor that blocks the interaction of human PD-1 with one of its ligands. In one embodiment, the second binding molecule blocks the interaction of PD-1 with PD-L1. In one embodiment, the second binding molecule blocks the interaction of PD-1 with PD-L2. In one embodiment, the second binding molecule blocks the interaction of PD-1 with PD-L1 and PD-L2. The order of the first and second binding molecule is not particularly limited and can be reversed.

In one embodiment, the PD-1 inhibitor is a V_(H) single domain antibody. Thus, another aspect relates to a binding molecule has the following formula: V_(H)(A)-L-V_(H)(B) wherein V_(H)(A)-is a V_(H) single domain antibody as disclosed herein and wherein V_(H)(B) is a V_(H) single domain antibody that blocks binding of PD-1 to PD-L1 and/or PD-L2. L is a linker. Suitable linkers include for example a linker with GS residues such as (Gly₄Ser)n, where n=from 1 to 20, e.g., 1 to 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, the linker is (Gly₄Ser)n, where n=4 or more. In another embodiment, the orientation is V_(H)(B)-L-V_(H)(A).

As demonstrated herein, we have surprisingly shown that a binding molecule has the following formula: V_(H)(A)-L-V_(H)(B) wherein V_(H)(A) is a blocking molecule and B a non blocking molecule as described above provides an enhanced effect compared to a V_(H) single domain antibody that blocks binding of PD-1 to PD-L1 and/or PD-L2 and is not linked to a non-blocking V_(H) single domain antibody. The effect is 10 to 25 fold compare d to a monovalent blocker. Thus, the V_(H) single domain antibodies described herein find particular use for combination with a V_(H) single domain antibody that does not block binding of PD-1 to PD-L1 and/or PD-L2 in a biparatopic molecule.

In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody chosen from Nivolumab®, Pembrolizumab® or Pidilizumab®. In some embodiments, the anti-PD-1 antibody is Nivolumab®. Alternative names for Nivolumab® include MDX-1106, MDX-1106-04, ONO-4538, or BMS-936558. In other embodiments, the anti-PD-1 antibody is Pembrolizumab®. Pembrolizumab® (Trade name KEYTRUDA® formerly Lambrolizumab®, also known as Merck 3745, MK-3475 or SCH-900475) is a humanized IgG4 monoclonal antibody that binds to PD-1. In some embodiments, the anti-PD-1 antibody is Pidilizumab®. Pidilizumab® (CT-011; Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD-1.

In one embodiment, the PD-1 inhibitor is a V_(H) single domain antibody.

In another embodiment, the second binding molecule is a PD-1 inhibitor that binds to PD-1, but does not block the interaction of human PD-1 with one of its ligands. Thus, the invention also relates to the use of single domain antibodies of the invention in a biparatopic construct together with a molecule, for example a single domain antibody that binds to PD-1 but does not block the functional interaction of PD-1 and its ligands.

In one embodiment, the binding molecule is multivalent, for example bivalent. Bivalent binding molecules comprise two V_(H) single domain antibodies that bind to the same target protein; e.g. human PD-1, at the same sites. In one embodiment, such molecules may comprise the same Humabody® V_(H). In another embodiment, such molecules may comprise two V_(H) single domain antibodies that are part of the same family, i.e. selected from the sequences shown in Table 1. In another embodiment, such molecules may comprise two V_(H) single domain antibodies that are not part of the same family, but bind to the same site on human PD-1.

Biparatopic and bivalent binding molecules of the present invention can be constructed using methods known in the art.

In certain embodiments, the binding agent is in the form of a multispecific, for example bispecific, binding agent providing multiple functionalities. Such multispecific agent comprises a single domain antibody according to the invention that has a first binding specificity to PD-1 and at least one further binding molecule with a second binding specificity. Said further binding molecule can be selected from an antibody, an antibody fragment or antibody mimetic. In one embodiment, said antibody fragment is selected from F(ab′)2, Fab, Fv, sFv or domain antibody. In one embodiment, said antibody fragment is a V_(H) single domain antibody.

In one embodiment, the binding agent is bispecific and comprises a single domain antibody according to the invention that has a first binding specificity to PD-1 and a second binding molecule with a second binding specificity. In one embodiment, the second binding molecule binds to an immunomodulatory agent, a checkpoint modulator, an agent involved in T-cell activation, a tumor microenvironment modifier (TME) or a tumour-specific target.

For example, the immunomodulator can be an inhibitor of an immune checkpoint molecule selected from an inhibitor of one or more of PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, CEACAM, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFR beta. In another embodiment, the immunomodulator can be an activator of a costimulatory molecule selected from an agonist of one or more of IL-2, IL-12, OX40, OX40L, CD2, CD3, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, B7-H4 or CD83 ligand, CD3, CD8, CD28, CD4 or ICAM-1. In one embodiment, the immunomodulator is an inhibitor of LAG-3.

In one embodiment, the binding agent described above comprises further binding molecules. Thus, the binding agent can be trispecific or tetraspecific. Additional specificities are also envisaged. Any combination of the aforesaid molecules can be made in a multispecific binding agent, for example, a trispecific binding agent that includes a single domain antibody of the invention and a second and third binding specificity.

In another embodiment, the at least second moiety may serve to prolong the half-life of the binding molecule. The second moiety may comprise a protein, for example an antibody, or part thereof that binds a serum albumin, e.g., human serum albumin (HSA) or mouse serum albumin (MSA). The second moiety may comprise a V_(H) domain that binds serum albumin, e.g., human serum albumin (HSA) or mouse serum albumin (MSA).

The second moiety may comprise a serum albumin, e.g. a human serum albumin (HSA) or a variant thereof such as HSA C34S. Further provided is binding molecule as described herein comprising a V_(H) domain and an Fc domain, e.g., wherein the V_(H) domain is fused to an Fc domain. Further provided is a binding molecule that comprises a second variable domain that specifically binds a second antigen, where the second antigen is an antigen other than human PD-1. The second antigen may be a cluster of differentiation (CD) molecule or a Major Histocompatibility Complex (MHC) Class II molecule.

In one embodiment, the anti-PD-1 single domain antibodies or multivalent binding agents of the invention are labelled with a detectable or functional label. A label can be any molecule that produces or can be induced to produce a signal, including but not limited to fluorophores, fluorescers, radiolabels, enzymes, chemiluminescers, a nuclear magnetic resonance active label or photosensitizers. Thus, the binding may be detected and/or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity or light absorbance.

In still other embodiments, the anti-PD-1 single domain antibodies or multivalent binding agents of the invention are coupled to at least one therapeutic moiety, such as a drug, an enzyme or a toxin. In one embodiment, the therapeutic moiety is a toxin, for example a cytotoxic radionuclide, chemical toxin or protein toxin.

In another aspect, the anti-PD-1 single domain antibodies or multivalent binding agents of the invention are modified to increase half-life, for example by a chemical modification, especially by PEGylation, or by incorporation in a liposome or using a serum albumin protein.

Half-life may be increased by at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding V_(H) single domain antibodies of the invention. For example, increased half-life may be more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding V_(H) single domain antibodies of the invention.

To generate a multivalent binding agent as described above, two binding molecules are connected by a linker, for example a polypeptide linker. Suitable linkers include for example a linker with GS residues such as (Gly₄Ser)n, where n=from 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

A single domain antibody described herein can be obtained from a transgenic rodent that expresses heavy chain only antibodies upon stimulation with a PD-1 antigen. The transgenic rodent, for example a mouse, preferably has a reduced capacity to express endogenous antibody genes. Thus, in one embodiment, the rodent has a reduced capacity to express endogenous light and/or heavy chain antibody genes. The rodent may therefore comprise modifications to disrupt expression of endogenous kappa and lambda light and/or heavy chain antibody genes so that no functional light and/or heavy chains are produced, for example as further explained below.

The invention also relates to a method for producing a human heavy chain only antibodies capable of binding human PD-1 said method comprising

-   -   a) immunising a transgenic rodent with an PD-1 antigen wherein         said rodent expresses a nucleic acid construct comprising         unrearranged human heavy chain V genes and is not capable of         making functional endogenous light or heavy chains,     -   b) isolating human heavy chain only antibodies.

Further steps can include isolating a V_(H) domain form said heavy chain only antibody, for example by generating a library of sequences comprising V_(H) domain sequences from said mouse and isolating sequences comprising V_(H) domain sequences from said libraries.

The invention also relates to a method for producing a single V_(H) domain antibody capable of binding human PD-1 said method comprising

-   -   a) immunising a transgenic rodent with an PD-1 antigen wherein         said rodent expresses a nucleic acid construct comprising         unrearranged human heavy chain V genes and is not capable of         making functional endogenous light or heavy chains,     -   b) generating a library of sequences comprising V_(H) domain         sequences from said mouse and     -   c) isolating sequences comprising V_(H) domain sequences from         said libraries.

Further steps may include identifying a single V_(H) domain antibody or heavy chain only antibody that binds to human PD-1 and blocks the interaction of PD-1 and PD-L1 and isolating said antibody, for example by using functional assays as shown in the examples.

Methods for preparing or generating the polypeptides, nucleic acids, host cells, products and compositions described herein using in vitro expression libraries can comprise the steps of:

-   -   a) providing a set, collection or library of nucleic acid         sequences encoding amino acid sequences; and     -   b) screening said set, collection or library for amino acid         sequences that can bind to/have affinity for PD-1 and     -   c) isolating the amino acid sequence(s) that can bind to/have         affinity for PD-1.

In the above method, the set, collection or library of amino acid sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) amino acid sequences will be clear to the person skilled in the art (see for example Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press; 1st edition (Oct. 28, 1996) Brian K. Kay, Jill Winter, John McCafferty).

Libraries, for example phage libraries, are generated by isolating a cell or tissue expressing an antigen-specific, heavy chain-only antibody, cloning the sequence encoding the V_(H) domain(s) from mRNA derived from the isolated cell or tissue and displaying the encoded protein using a library. The V_(H) domain(s) can be expressed in bacterial, yeast or other expression systems.

The invention also relates to an isolated V_(H) single domain antibody or an isolated heavy chain only antibody comprising a V_(H) domain binding to PD-1 comprising an amino acid product of or derived from a human V_(H) germline sequence. The heavy chain only antibody may be fully human or comprise mouse sequences.

In the various aspects and embodiments of the invention as out herein, the term rodent may relate to a mouse or a rat.

In one embodiment, the rodent is a mouse. The mouse may comprise a non-functional endogenous lambda light chain locus. Thus, the mouse does not make a functional endogenous lambda light chain. In one embodiment, the lambda light chain locus is deleted in part or completely or rendered non-functional through insertion, inversion, a recombination event, gene editing or gene silencing. For example, at least the constant region genes C1, C2 and C3 may be deleted or rendered non-functional through insertion or other modification as described above. In one embodiment, the locus is functionally silenced so that the mouse does not make a functional lambda light chain.

Furthermore, the mouse may comprise a non-functional endogenous kappa light chain locus. Thus, the mouse does not make a functional endogenous kappa light chain. In one embodiment, the kappa light chain locus is deleted in part or completely or rendered non-functional through insertion, inversion, a recombination event, gene editing or gene silencing. In one embodiment, the locus is functionally silenced so that the mouse does not make a functional kappa light chain.

The mouse having functionally-silenced endogenous lambda and kappa L-chain loci may, for example, be made as disclosed in WO 2003/000737, which is hereby incorporated by reference in its entirety.

Furthermore, the mouse may comprise a non-functional endogenous heavy chain locus. Thus, the mouse does not make a functional endogenous heavy chain. In one embodiment, the heavy chain locus is deleted in part or completely or rendered non-functional through insertion, inversion, a recombination event, gene editing or gene silencing. In one embodiment, the locus is functionally silenced so that the mouse does not make a functional heavy chain.

For example, as described in WO 2004/076618 (hereby incorporated by reference in its entirety), all 8 endogenous heavy chain constant region immunoglobulin genes (μ, δ, γ3, γ1, γ2a, γ2b, ε and α) are absent in the mouse, or partially absent to the extent that they are non-functional, or genes δ, γ3, γ1, γ2a, γ2b and ε are absent and the flanking genes μ and α are partially absent to the extent that they are rendered non-functional, or genes μ, δ, γ3, γ1, γ2a, γ2b and ε are absent and a is partially absent to the extent that it is rendered non-functional, or δ, γ3, γ1, γ2a, γ2b, ε and a are absent and μ is partially absent to the extent that it is rendered non-functional. By deletion in part is meant that the endogenous locus gene sequence has been deleted or disrupted, for example by an insertion, to the extent that no functional endogenous gene product is encoded by the locus, i.e., that no functional product is expressed from the locus. In another embodiment, the locus is functionally silenced.

In one embodiment, the mouse comprises a non-functional endogenous heavy chain locus, a non-functional endogenous lambda light chain locus and a non-functional endogenous kappa light chain locus. The mouse therefore does not produce any functional endogenous light or heavy chains. Thus, the mouse is a triple knockout (TKO) mouse.

The transgenic mouse may comprise a vector, for example a Yeast Artificial Chromosome (YAC) for expressing a heterologous, preferably a human, heavy chain locus. YACs are vectors that can be employed for the cloning of very large DNA inserts in yeast. As well as comprising all three cis-acting structural elements essential for behaving like natural yeast chromosomes (an autonomously replicating sequence (ARS), a centromere (CEN) and two telomeres (TEL)), their capacity to accept large DNA inserts enables them to reach the minimum size (150 kb) required for chromosome-like stability and for fidelity of transmission in yeast cells. The construction and use of YACs is well known in the art (e.g., Bruschi, C. V. and Gjuracic, K. Yeast Artificial Chromosomes, Encyclopedia of Life Sciences, 2002 Macmillan Publishers Ltd, Nature Publishing Group).

For example, the YAC may comprise a plethora of unrearranaged human V_(H), D and J genes in combination with mouse immunoglobulin constant region genes lacking C_(H)1 domains, mouse enhancer and regulatory regions. The human V_(H), D and J genes are human V_(H), D and J loci and they are unrearranged genes that are fully human. An example of such a YAC is provided in the example section.

Alternative methods known in the art may be used for deletion or inactivation of endogenous mouse or rat immunoglobulin genes and introduction of human V_(H), D and J genes in combination with mouse immunoglobulin constant region genes lacking C_(H)1 domains, mouse enhancer and regulatory regions.

Transgenic mice can be created according to standard techniques as illustrated in the examples. The two most characterised routes for creating transgenic mice are via pronuclear microinjection of genetic material into freshly fertilised oocytes or via the introduction of stably transfected embryonic stem cells into morula or blastocyst stage embryos.

Regardless of how the genetic material is introduced, the manipulated embryos are transferred to pseudo-pregnant female recipients where pregnancy continues and candidate transgenic pups are born.

The main differences between these broad methods are that ES clones can be screened extensively before their use to create a transgenic animal. In contrast, pronuclear microinjection relies on the genetic material integrating to the host genome after its introduction and, generally speaking, the successful incorporation of the transgene cannot be confirmed until after pups are born.

There are many methods known in the art to both assist with and determine whether successful integration of transgenes occurs. Transgenic animals can be generated by multiple means including random integration of the construct into the genome, site-specific integration, or homologous recombination. There are various tools and techniques that can be used to both drive and select for transgene integration and subsequent modification including the use of drug resistance markers (positive selection), recombinases, recombination-mediated cassette exchange, negative selection techniques, and nucleases to improve the efficiency of recombination. Most of these methods are commonly used in the modification of ES cells. However, some of the techniques may have utility for enhancing transgenesis mediated via pronuclear injection.

Further refinements can be used to give more efficient generation of the transgenic line within the desired background. As described above, in preferred embodiments, the endogenous mouse immunoglobulin expression is silenced to permit sole use of the introduced transgene for the expression of the heavy-chain only repertoire that can be exploited for drug discovery. Genetically-manipulated mice, for example TKO mice that are silenced for all endogenous immunoglobulin loci (mouse heavy chain, mouse kappa chain and mouse lambda chain) can be used as described above. The transfer of any introduced transgene to this TKO background can be achieved via breeding, either conventional or with the inclusion of an IVF step to give efficient scaling of the process. However, it is also possible to include the TKO background during the transgenesis procedure. For example, for microinjection, the oocytes may be derived from TKO donors. Similarly, ES cells from TKO embryos can be derived for use in transgenesis.

Triple knock-out mice into which transgenes have been introduced to express immunoglobulin loci are referred to herein as TKO/Tg.

In one embodiment, the mouse is as described in WO2016/062990.

The invention also relates to a rodent, preferably a mouse which expresses a human heavy chain locus and which has been immunized with a PD-1 antigen. The invention also relates to a rodent as described above, preferably a mouse which expresses a heavy chain only antibody comprising a human V_(H) domain that binds to human PD-1. Preferably, said rodent is not capable of making functional endogenous kappa and lambda light and/or heavy chains. The human heavy chain locus is located on a transgene which can be as described above.

The invention also relates to an anti-human PD-1 single V_(H) domain antibody or an anti-human PD-1 heavy chain only antibody comprising a human V_(H) domain or obtained or obtainable from a rodent, preferably a mouse, immunised with a human PD-1 antigen and which expresses a human heavy chain locus. Preferably, said rodent is not capable of making functional endogenous kappa and lambda light and/or heavy chains. The human heavy chain locus is located on a transgene which can be as described above.

In another aspect of the present invention, there is provided a pharmaceutical composition comprising a single domain antibody according to the present invention and optionally a pharmaceutically acceptable carrier. A single domain antibody of the present invention or the pharmaceutical composition of the invention can be administered by any convenient route, including but not limited to oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intranasal, pulmonary, intradermal, intravitrial, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal, by inhalation, or topical, particularly to the ears, nose, eyes, or skin or by inhalation.

Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical or subcutaneous administration. Preferably, the compositions are administered parenterally.

The pharmaceutically acceptable carrier or vehicle can be particulate, so that the compositions are, for example, in tablet or powder form. The term “carrier” refers to a diluent, adjuvant or excipient, with which a drug antibody conjugate of the present invention is administered. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. In one embodiment, when administered to an animal, the single domain antibody of the present invention or compositions and pharmaceutically acceptable carriers are sterile. Water is a preferred carrier when the drug antibody conjugates of the present invention are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

The pharmaceutical composition of the invention can be in the form of a liquid, e.g., a solution, emulsion or suspension. The liquid can be useful for delivery by injection, infusion (e.g., IV infusion) or sub-cutaneously.

When intended for oral administration, the composition is preferably in solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.

As a solid composition for oral administration, the composition can be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition typically contains one or more inert diluents. In addition, one or more of the following can be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, corn starch and the like; lubricants such as magnesium stearate; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent. When the composition is in the form of a capsule (e. g. a gelatin capsule), it can contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.

The composition can be in the form of a liquid, e. g. an elixir, syrup, solution, emulsion or suspension. The liquid can be useful for oral administration or for delivery by injection. When intended for oral administration, a composition can comprise one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition for administration by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can also be included.

Compositions can take the form of one or more dosage units.

In specific embodiments, it can be desirable to administer the composition locally to the area in need of treatment, or by intravenous injection or infusion.

The amount of the single domain antibody of the present invention that is effective/active in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account.

Typically, the amount is at least about 0.01% of a single domain antibody of the present invention by weight of the composition. When intended for oral administration, this amount can be varied to range from about 0.1% to about 80% by weight of the composition. Preferred oral compositions can comprise from about 4% to about 50% of the sdAb of the present invention by weight of the composition.

Preferred compositions of the present invention are prepared so that a parenteral dosage unit contains from about 0.01% to about 2% by weight of the single domain antibody of the present invention.

For administration by injection, the composition can comprise from about typically about 0.1 mg/kg to about 250 mg/kg of the animal's body weight, preferably, between about 0.1 mg/kg and about 20 mg/kg of the animal's body weight, and more preferably about 1 mg/kg to about 10 mg/kg of the animal's body weight. In one embodiment, the composition is administered at a dose of about 1 to 30 mg/kg, e.g., about 5 to 25 mg/kg, about 10 to 20 mg/kg, about 1 to 5 mg/kg, or about 3 mg/kg. The dosing schedule can vary from e.g., once a week to once every 2, 3, or 4 weeks.

The invention provides methods of treating PD-1-mediated diseases or disorders in a mammal, e.g., a human patient, comprising administering an effective amount of an antibody of the present invention to a mammal in need thereof. In particular, the invention furthermore relates to a method for the prevention and/or treatment of a disorder selected from cancer, an immune disorder, neurological disease, inflammatory disorder, allergy, transplant rejection, viral infection, immune deficiency, and other immune system-related disorder said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of a single domain antibody or pharmaceutical composition of the invention, or of a pharmaceutical composition of the invention.

As used herein, “treat”, “treating” or “treatment” means inhibiting or relieving a disease or disorder. For example, treatment can include a postponement of development of the symptoms associated with a disease or disorder, and/or a reduction in the severity of such symptoms that will, or are expected, to develop with said disease. The terms include ameliorating existing symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, the terms denote that a beneficial result is being conferred on at least some of the mammals, e.g., human patients, being treated. Many medical treatments are effective for some, but not all, patients that undergo the treatment.

The term “subject” or “patient” refers to an animal which is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, murine, bovine, equine, canine, ovine, or feline.

As used herein, the term “effective amount” means an amount of an anti-PD-1 antibody, that when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject, is effective to achieve the desired therapeutic or prophylactic effect under the conditions of administration

The invention also relates to a single domain antibody or pharmaceutical composition of the invention for use in the treatment or prevention of a disease.

In another aspect, the invention relates to a single domain antibody or pharmaceutical composition of the invention for use in the treatment or prevention of cancer, an immune disorder, neurological disease, inflammatory disorder, allergy, transplant rejection, viral infection, immune deficiency, and other immune system-related disorder.

In another aspect, the invention relates to the use of a single domain antibody or pharmaceutical composition of the invention in the treatment or prevention of a disease.

In another aspect, the invention relates to the use of a single domain antibody or pharmaceutical composition of the invention in the manufacture of a medicament for the treatment or prevention of cancer, an immune disorder, neurological disease, inflammatory disorder, allergy, transplant rejection, viral infection, immune deficiency, and other immune system-related disorder.

The cancer can be selected from a solid or non-solid tumor. For example, the cancer may be selected from bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, breast cancer, brain cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, kidney cancer, sarcoma of soft tissue, cancer of the urethra, cancer of the bladder, renal cancer, lung cancer, non-small cell lung cancer, thymoma, urothelial carcinoma leukemia, prostate cancer, mesothelioma, adrenocortical carcinoma, lymphomas, such as such as Hodgkin's disease, non-Hodgkin's, gastric cancer, and multiple myelomas.

In one embodiment, the tumor is a solid tumor. Examples of solid tumors which may be accordingly treated include breast carcinoma, lung carcinoma, colorectal carcinoma, pancreatic carcinoma, glioma and lymphoma. Some examples of such tumors include epidermoid tumors, squamous tumors, such as head and neck tumors, colorectal tumors, prostate tumors, breast tumors, lung tumors, including small cell and non-small cell lung tumors, pancreatic tumors, thyroid tumors, ovarian tumors, and liver tumors. Other examples include Kaposi's sarcoma, CNS, neoplasms, neuroblastomas, capillary hemangioblastomas, meningiomas and cerebral metastases, melanoma, gastrointestinal and renal carcinomas and sarcomas, rhabdomyosarcoma, glioblastoma, preferably glioblastoma multiforme, and leiomyosarcoma. Examples of vascularized skin cancers for which the antagonists of this invention are effective include squamous cell carcinoma, basal cell carcinoma and skin cancers that can be treated by suppressing the growth of malignant keratinocytes, such as human malignant keratinocytes.

In one embodiment, the tumor is a non-solid tumor. Examples of non-solid tumors include leukemia, multiple myeloma and lymphoma.

In one aspect, the cancer is identified as a PD-L1 positive cancer. In one aspect, the cancer is locally advanced unresectable, metastatic, or recurrent cancer.

Preferred cancers whose growth may be inhibited using the antibodies of the invention include cancers typically responsive to immunotherapy. Non-limiting examples of preferred cancers for treatment include melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g. clear cell carcinoma), prostate cancer (e.g. hormone refractory prostate adenocarcinoma), breast cancer, colon cancer and lung cancer (e.g. non-small cell lung cancer).

In one embodiment, the cancer has progressed after another treatment, for example chemotherapy.

The single domain antibodies and pharmaceutical compositions of the present invention are particularly useful for the treatment of cancers that are associated with cells (e.g., exhausted T cells, B cells, monocytes, etc.) that express abnormally high levels of PD-1. Other preferred cancers include those characterized by elevated expression of PD-1 and/or its ligands PD-L1 and/or PD-L2. In one embodiment, the cancer is selected from a cancer that has high levels of cancer-associated genetic mutations and/or high levels of expression of tumour antigens. In another embodiment, the cancer is selected from a cancer known to be immunogenic or that is able to become immunogenic upon treatment with other cancer therapies.

The immune disorder can be selected from graft vs. host disease, arthritis, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erthematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, Neuromyelitis optica (NMO), type 1 or immune-mediated diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus, lupus erythematosus, takayasu arteritis, temporal arteristis/giant cell arteritis, transverse myelitis, ulcerative colitis, uveitis, vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, and Wegener's granulomatosis.

The neurological disease can be selected from Alzheimer's disease, epilepsy, Parkinson's disease, dementia, multiple sclerosis, peripheral neuropathy or post-herpetic neuralgia.

The single domain antibody or pharmaceutical composition of the invention may be administered as the sole active ingredient or in combination with one or more other therapeutic agent. A therapeutic agent is a compound or molecule which is useful in the treatment of a disease. Examples of therapeutic agents include antibodies, antibody fragments, drugs, toxins, nucleases, hormones, immunomodulators, pro-apoptotic agents, anti-angiogenic agents, boron compounds, photoactive agents or dyes and radioisotopes. An antibody molecule includes a full antibody or fragment thereof (e.g., a Fab, F(ab′)2, Fv, a single chain Fv fragment (scFv) or a single domain antibody, for example a V_(H) domain, or antibody mimetic protein.

In one embodiment, the single domain antibody is used in combination with an existing therapy or therapeutic agent, for example an anti-cancer therapy. Thus, in another aspect, the invention also relates to a combination therapy comprising administration of a single domain antibody or pharmaceutical composition of the invention and an anti-cancer therapy. The anti-cancer therapy may include a therapeutic agent or radiation therapy and includes gene therapy, viral therapy, RNA therapy bone marrow transplantation, nanotherapy, targeted anti-cancer therapies or oncolytic drugs. Examples of other therapeutic agents include other checkpoint inhibitors, antineoplastic agents, immunogenic agents, attenuated cancerous cells, tumor antigens, antigen presenting cells such as dendritic cells pulsed with tumor-derived antigen or nucleic acids, immune stimulating cytokines (e.g., IL-2, IFNa2, GM-CSF), targeted small molecules and biological molecules (such as components of signal transduction pathways, e.g. modulators of tyrosine kinases and inhibitors of receptor tyrosine kinases, and agents that bind to tumor-specific antigens, including EGFR antagonists), an anti-inflammatory agent, a cytotoxic agent, a radiotoxic agent, or an immunosuppressive agent and cells transfected with a gene encoding an immune stimulating cytokine (e.g., GM-CSF), chemotherapy. In one embodiment, the single domain antibody is used in combination with surgery.

In one embodiment, the single domain antibody or pharmaceutical composition of the invention is administered together with an immunomodulator, a checkpoint modulator, an agent involved in T-cell activation, a tumor microenvironment modifier (TME) or a tumour-specific target. For example, the immunomodulator can be an inhibitor of an immune checkpoint molecule selected from an inhibitor of one or more of PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, CEACAM, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFR beta. In another embodiment, the immunomodulator can be an activator of a costimulatory molecule selected from an agonist of one or more of OX40, OX40L, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83 ligand, CD3, CD8, CD28, CD4 or ICAM-1.

In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody chosen from Nivolumab®, Pembrolizumab® or Pidilizumab®.

In a specific embodiment of the present invention, the composition is administered concurrently with a chemotherapeutic agent or with radiation therapy. In another specific embodiment, the chemotherapeutic agent or radiation therapy is administered prior or subsequent to administration of the composition of the present invention, preferably at least an hour, five hours, 12 hours, a day, a week, a month, more preferably several months (e. g. up to three months), prior or subsequent to administration of composition of the present invention.

In some embodiments, the single domain antibodies of the invention may be administered with two or more therapeutic agents. In some embodiments, the binding agents of the invention may be administered with two or more therapeutic agents.

The single domain antibody or pharmaceutical composition of the invention may be administered at the same time or at a different time as the other therapy or therapeutic compound or therapy, e.g., simultaneously, separately or sequentially.

In yet another aspect, the invention provides a method of modulating an immune response in a subject comprising administering to the subject the single domain antibody of the invention such that the immune response in the subject is modulated. Preferably, the antibody of the invention enhances, stimulates or increases the immune response in the subject.

In a further aspect, the invention provides a method of inhibiting growth of tumor cells in a subject, comprising administering to a subject a therapeutically effective amount of an anti-PD-1 single domain antibody of the invention.

In another aspect, the invention relates to an immunoconjugate comprising a single domain antibody of the invention conjugated to at least one therapeutic and/or diagnostic agent.

In another aspect, the invention provides a kit for the treatment or prevention of a disease or an immune response and/or for detecting PD-1 for diagnosis, prognosis or monitoring disease comprising a single domain antibody of the invention. Such a kit may contain other components, packaging, instructions, or material to aid in the detection of PD-1 protein. The kit may include a labeled single domain antibody of the invention as described above and one or more compounds for detecting the label.

The invention in another aspect provides a single domain antibody of the invention packaged in lyophilized form, or packaged in an aqueous medium.

The invention also relates to a single domain antibody as described herein with reference to the figures and examples.

In another aspect, single domain antibodies of the invention are used for non-therapeutic purposes, such as diagnostic tests and assays. A method for detecting the presence of human PD-1 in a test sample comprises contacting said sample with a single domain antibody according to the invention and at least one detectable label and detecting binding of said single domain antibody to human PD-1.

In one embodiment, the invention relates to a method of diagnosing a PD-1-mediated adaptive immune resistance in a patient who has cancer. The method comprises contacting a sample with a single domain antibody disclosed herein that has been labelled with a detectable moiety; and detecting expression of PD-1 on immune cells, e.g., CD8+ T cells; B cells; and macrophages. The sample may be tumor tissue.

Modifications of antibodies for diagnostic purposes are well known in the art. For example, antibodies may be modified with a ligand group such as biotin, or a detectable marker group such as a fluorescent group, a radioisotope, or an enzyme. Compounds of the invention can be labelled using conventional techniques. Suitable detectable labels include but are not limited to fluorophores, chromophores, radioactive atoms, electron-dense reagents, enzymes, and ligands having specific binding partners.

Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods, as well as the best mode thereof, of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

All documents mentioned in this specification are incorporated herein by reference in their entirety, including references to gene accession numbers and references to patent publications.

“and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

The invention is further described in the non-limiting examples.

EXAMPLES Example 1. Construction of Tg/TKO Mice

Mice carrying a human heavy-chain antibody transgenic locus in germline configuration within a background that is silenced for endogenous heavy and light chain antibody expression (triple knock-out, or TKO) were created as previously described (WO2004/076618, WO2003/000737, Ren et al., Genomics, 84, 686, 2004; Zou et al., J. Immunol., 170, 1354, 2003 and WO2016/062990). Briefly, transgenic mice were derived following pronuclear microinjection of freshly fertilised oocytes with a yeast artificial chromosome (YAC) comprising a plethora of human V_(H), D and J genes in combination with mouse immunoglobulin constant region genes lacking C_(H)1 domains, mouse enhancer and regulatory regions. Yeast artificial chromosomes (YACs) are vectors that can be employed for the cloning of very large DNA inserts in yeast. As well as comprising all three cis-acting structural elements essential for behaving like natural yeast chromosomes (an autonomously replicating sequence (ARS), a centromere (CEN) and two telomeres (TEL)), their capacity to accept large DNA inserts enables them to reach the minimum size (150 kb) required for chromosome-like stability and for fidelity of transmission in yeast cells. The construction and use of YACs is well known in the art (e.g., Bruschi, C. V. and Gjuracic, K. Yeast Artificial Chromosomes, Encyclopedia of Life Sciences, 2002, Macmillan Publishers Ltd., Nature Publishing Group).

The YAC used comprised multiple human heavy chain V genes, multiple human heavy chain D and J genes, a murine C_(H)1 gene and a murine 3′ enhancer gene. It lacks the C_(H)1 exon.

The transgenic founder mice were back crossed with animals that lacked endogenous immunoglobulin expression to create the Tg/TKO lines used in the immunisation studies described.

Example 2. Antigen for Immunisation

The immunisations used recombinant human PD-1 Fc chimera purchased from R&D, catalogue number 1086-PD, lot number FVQ081502B or FVQ081503A.

Recombinant human PD-1-TetTox protein was used in another separate immunisation. This is based on residues 1-167 of human PD-1 and comprises at the N terminus the tet toxin linked to PD-1 via a polylinker. Also included is an N-terminal His tag as well as leader sequence and restriction site for proteolytic cleavage.

CHO cell lines expressing human PD-1 on the surface were made in house.

Example 3. Immunisation Protocol

Tg/TKO mice aged 8-12 weeks of age each received an initial prime dose of either 50 ug or 10 ug of recombinant purified human PD-1-Fc protein emulsified in Complete Freund's Adjuvant and delivered subcutaneously, followed by three boosts of 10 μg of the recombinant protein, emulsified in Incomplete Freund's Adjuvant, also administered subcutaneously, given at various intervals following the initial priming. A final dose of 10 μg or 20 ug recombinant purified human PD-1 protein antigen was administered intraperitoneally, in phosphate buffered saline, in the absence of adjuvant.

A separate cohort of Tg/TKO mice aged 8-12 weeks of age each received an initial prime dose of 10 ug of recombinant purified human PD1-TetTox protein emulsified in Complete Freund's Adjuvant and delivered subcutaneously, followed by three boosts of 10 μg emulsified in Incomplete Freund's Adjuvant, also administered subcutaneously, given at various intervals following the initial priming. A final dose of 10 μg recombinant purified human PD-1-Tettox protein antigen was administered intraperitoneally, in phosphate buffered saline, in the absence of adjuvant.

Another cohort of animals was primed with 50 ug purified human PD-1-Fc protein as above, followed by three boosts of 10 million cells expressing human PD-1 at high levels on the surface. The boosts were given without adjuvant, two of them sub cutaneously and the third intraperitoneally. A final dose of 10 μg recombinant purified human PD-1-Fc protein was administered intraperitoneally, in phosphate buffered saline, in the absence of adjuvant.

Example 4. Serum ELISA

Serum was collected from mice before and after immunisation, and checked by ELISA for the presence of serum PD-1/Fc reactive heavy chain antibodies in response to immunisation with PD-1 antigen. Nunc Maxisorp plates (Nunc cat. no. 443404) were coated overnight at 4° C. with 50 μl/well of either a 1 μg/ml recombinant huPD-1-Fc solution in PBS (R&D 1086-PD) or of hPD-1 HIS in PBS R&D (8986-PD or 9047-PD). Plates were washed using PBS (prepared from PBS tablets, Oxoid cat. no. BR0014G) supplemented with 0.05% (v/v) Tween® 20 (Sigma P1379), followed by washes with PBS without added Tween 20. To block non-specific protein interactions, a solution of 3% (w/v) skimmed milk powder (Marvel®) in PBS was added to the wells and the plate was incubated for at least one hour at room temperature, then discarded.

Whole blood samples were centrifuged at 13000 rpm for 5 mins to separate blood from serum. Dilutions of serum were prepared in 3% Marvel®/PBS in polypropylene tubes or plates, pre-incubated for at least one hour at room temperature then transferred to the blocked ELISA plate and incubated for at least one hour. Unbound protein was removed by repetitive washing with PBS/Tween 20 followed by PBS. A 1:10,000 solution of biotin-conjugated, goat anti-mouse IgG, Fcgamma subclass 1 specific antibody (Jackson cat. no. 115-065-205), prepared in PBS/3% Marvel® was added to each well and incubated at room temperature for at least one hour. Unbound detection antibody was removed by repeated washing using PBS/Tween® 20 and PBS. Neutravidin-HRP solution (Pierce cat. no. 31030) in 3% Marvel®/PBS was added to the ELISA plates and allowed to bind for 30 minutes, then washed as above. The ELISA was developed using TMB substrate (Sigma cat. no. T0440) and the reaction was stopped after 7 minutes by the addition of 50 ul 0.5M H2504 solution (Sigma cat. no. 320501). Absorbances were read at 450 nm with the BMG Pherastar.

Mice were checked by ELISA for the presence of antibody in serum. All mice showed a robust immune response.

Example 5. Generation of Libraries from Immunised Mice Tissue Collection and Homogenisation

Generation of libraries from immunised mice described above followed standard protocols of library generation as summarised below.

Total spleen, inguinal and brachial lymph nodes were used according to standard protocols.

RNA Extraction and RT-PCR

Spleen: 400 μl supernatant was used for preparation of total RNA. RNA was extracted using Qiagen RNeasy® kit (cat. no. 74104) following the manufacturer's protocol.

Lymph nodes: prepared by essentially the same process on the Kingfisher.

V_(H) sequences were mined from the RNA samples using Superscript III RT-PCR high-fidelity kit (Invitrogen cat. no. 12574-035) according to the manufacturer's protocol. For each spleen and LN RNA sample, RT-PCR reactions were performed using a single JH primer in combination with primers for V_(H)1, V_(H)2, V_(H)3, V_(H)4 or V_(H)6 families.

RT-PCR products were pooled so that V_(H)1 products from lymph nodes 1˜4 and spleen were combined. Amplified material was purified using the GeneJet™ purification kit (cat #K0702) according to the manufacturer's protocol, eluting in 50 ul water.

Cloning into Phagemid Vector

The phagemid vector, pUCG3, was employed in these studies. A conventional PCR-based method was used to construct the V_(H) phagemid libraries from the amplified V_(H) sequences. In short, he following procedure was used:

A linearised version of pUCG3 was created using PCR. Vector PCR product (3152 bp) was gel purified using Fermentas GeneJet Gel purification kit (cat. no. K0691), according to the manufacturer's instructions. Purified V_(H) RT-PCR products were used to prime a PCR reaction from the linearised pUCG3 resulting in a heterogeneous population of V_(H) cloned into pUCG3.

PCR products were analysed on a 1% (w/v) agarose gel.

Generation of Phagemid Library

V_(H)/phagemid PCR products were pooled by animal-of-origin and purified using Fermentas PCR purification kit (cat. no. K0702) according to the manufacturer's instructions. The final elution was in 22 μl H₂O.

Eluted DNA was used to transform TG1 E. coli (Lucigen, cat. no. 60502-2) by electroporation using the Bio-Rad GenePulser Xcell pulsed at 2500V, 25 uF, 200W. Electroporated cells were pooled.

A 10-fold dilution series of the transformations was plated on 2×TY agar petri plates with 2% (w/v) glucose and 100 μg/ml ampicillin. Resulting colonies on these dishes were used to estimate library size. The remainder of the transformation was plated on large format 2×TY agar Bioassay dishes supplemented with 2% (w/v) glucose and 100 μg/ml ampicillin. All agar plates were incubated overnight at 30° C.

Libraries were harvested by adding 10 ml of 2×TY broth to the large format bioassay dishes. Bacterial colonies were gently scraped and OD600 recorded. Aliquots were stored at −80° C. in cryovials after addition of an equal volume of 50% (v/v) glycerol solution or used directly in a phage selection process.

Example 6. Selection Strategies for Isolation of PD-1 Binding V_(H) Isolation and Optimisation

Preparation of library phage stocks and phage display selections were performed according to published methods (Antibody Engineering, edited by Benny Lo, chapter 8, p161-176, 2004). In most cases, phage display combined with a panning approach was used to isolate binding V_(H) domains. However, a variety of different selection methods are well described in the art, including soluble selection and selections performed under stress (e.g., heat).

Standard methods were employed for optimisation, for example to increase V_(H) affinity for antigen. These optimisation strategies are described in the art:

-   -   a) shuffling (Antibody Engineering, Edited by Benny Lo, chapter         19, p327-343, 2004);     -   b) Targeted randomisation of CDR3 regions using randomised         oligonucleotides and phage display technology (Main et al., J         Pharmacol Exp Ther. 2006 December; 319(3): 1395-404.) and     -   c) Ribosome display.

Example 7: Screening of Periplasmic Extracts for Binding to CHO Human PD1 Cells and Inhibition of PD-L1 Binding PD-1

Following selections of the libraries, specific V_(H) that bound to CHO cells expressing human PD-1 and inhibited the interaction between recombinant human PD-1 protein and recombinant human PD-L1 protein were identified by single point screening of bacterial periplasmic extracts.

Small-scale bacterial periplasmic extracts were prepared from 1 ml cultures, grown in deep well plates. Starter cultures were used to inoculate 96-well deep well plates (Fisher, cat. no. MPA-600-030X) containing 2×TY broth (Melford cat. no. M2130), supplemented with 0.1% (w/v) glucose and 100 μg/ml ampicillin at 37° C. with 250 rpm shaking. When OD₆₀₀ had reached 0.6-1, V_(H) production was induced by adding 100 μl of 2×TY, supplemented with IPTG (final concentration 0.5 mM) and ampicillin and the cultures were grown overnight at 30° C. with shaking at 220 rpm. E. coli were pelleted by centrifugation at 3200 rpm for 10 mins and supernatants discarded. Cell pellets were resuspended in 120 μl of ice cold extraction buffer (50 mM MOPs, 0.5 mM EDTA, 0.5M Sucrose), then 180 μl of 1:5 diluted ice cold extraction buffer added. Cells were incubated on ice for 30 minutes and then centrifuged at 4500 rpm for 15 mins at 4° C. Supernatants were transferred to polypropylene plates for testing in assays.

Binding of His-tagged V_(H) in the supernatants to CHO cell expressed human PD-1 was assessed using Fluorescence Microvolume Assay Technology (FMAT), a fluorescence-based platform that detects fluorescence localized to beads or cells settled at the bottom of microwells (Dietz et al., Cytometry 23:177-186 (1996), Miraglia et al., J. Biomol. Screening 4:193-204 (1999). A CHO TREX human PD-1 cell line was generated in-house using full-length human PD-1 sequence by standard procedures. All reagents were prepared in FMAT assay buffer (pH 7.4) containing PBS, 0.1% Bovine Serum Albumin, 0.01% Sodium Azide. Peripreps were transferred into 384 well black clear-bottomed assay plates (Costar cat. no. 3655) and incubated for a minimum of 2 hours at room temperature with 1.5 nM Anti-His (Millipore cat. no. 05-949)/3 nM Goat Anti-Mouse Alexa Fluor-488 (Jackson Immunolabs cat. no. 115-545-071) and 2000 CHO human PD-1 cells prestained with DRAQ5 (Thermo Scientific cat. no. 62251). Plates were read in the FL2 (502 nm-537 nm) and FL5 (677-800 nm) channels on the TTP Mirrorball plate reader following excitation at 488 nm and 640 nm. Data was gated on FL5 perimeter and peak intensity and the FL2 median mean fluorescence intensity of the gated data used for determination of V_(H) binding.

In parallel to the CHO PD-1 binding assay, periplasmic extracts were tested for their ability to inhibit the interaction of PD-L1 protein with PD-1 protein by single point screening in an HTRF inhibition assay. All samples and reagents were prepared in HTRF assay buffer containing PBS, 0.1% (w/v) BSA and 0.4M Potassium Fluoride. Periplasmic extracts were incubated with 25 nM strep tagged PD-L1 (Acro Biosystems cat no. PD-1-H5282), 1.5 nM Anti human-Fc Cryptate PAb (Cisbio cat. no. 61HFCKLB), 10 nM StrepMAB-Oyster 645 conjugate in black 384-shallow-well plates (Costar cat. no. 3676) for a minimum of 3 hours at room temperature. Total binding controls containing periplasmic extract sample buffer and non-specific binding controls containing excess untagged competitor were set up on each plate for data normalisation. Time-resolved fluorescent emission at 620 nm and 665 nm was measured following excitation at 337 nm on the BMG PHERAstar plate reader. Data was expressed as a % of the total binding control (% control) after subtraction of the background signal determined from the non-specific binding control wells.

Families of V_(H) were identified that bound to the CHO human PD-1 cells with FL2 fluorescence >1000 (FIG. 1A) and that inhibited PD-1 binding to PD-L1 (FIG. 1B).

Example 8. Sequencing

Each individual V_(H) clone as identified above was sequenced from the phagemid and grouped based on V_(H) germline and CDR3 amino acid similarity. Representative clones were further characterised. Further clones were generated by sequence optimisation of clone 1.1 as shown in table 1. Standard methods were used for optimisation. Clones 1.1 to 1.40 isolated as above were grouped into a single family. Clones 1.41-1.115 are sequence optimised variants clone 1.1.

Example 9. Preparation and Characterisation of Purified V_(H) a) Preparation of Purified V_(H)

Purified V_(H) were obtained by using the V_(H) C-terminal 6×HIS tag for nickel-agarose affinity chromatographic purification of the periplasmic extracts. A starter culture of each V_(H) was grown overnight in 2×TY media (2×TY broth (Melford cat. no. M2103) supplemented with 2% (w/v) glucose+100 μg/ml ampicillin at 30° C. with 250 rpm shaking. This overnight culture was then used to inoculate 50 ml-200 ml 2×TY media and incubated at 37° C. with 250 rpm shaking for approximately 6-8 hours (until OD₆₀₀=0.6-1.0). Cultures were centrifuged at 3200 rpm for 10 mins and the cell pellets resuspended in fresh 2×TY broth containing 100 μg/ml ampicillin+1 mM IPTG. Shake flasks were incubated overnight at 30° C. and 250 rpm. Cultures were again centrifuged at 3200 rpm for 10 mins and supernatants discarded. Cell pellets were resuspended in ice cold extraction buffer (20% (w/v) sucrose, 1 mM EDTA, 50 mM Tris-HCl pH 8.0 or 50 mM MOPS) by gently pipetting then diluted further with 1:5 diluted ice cold extraction buffer. Cells were incubated on ice for 30 minutes then centrifuged at 4500 rpm for 15 mins at 4° C. Supernatants were transferred to tubes containing imidazole (Sigma cat. no. 12399—final concentration 10 mM) and pre-equilibrated nickel agarose beads (Qiagen, Ni-NTA 50% soln, cat. no. 30210). V_(H) binding was allowed to proceed for 2 hours at 4° C. with gentle shaking. The beads were transferred to a polyprep column (BioRad cat. no. 731-1550) and the supernatant discarded by gravity flow. Columns were washed 3 times with PBS+0.05% Tween® followed by 3 washes with 5 ml of PBS/20 mM Imidazole. V_(H) were eluted from the columns using PBS/250 mM imidazole. The imidazole was removed from the purified V_(H) preparations by buffer exchange with NAP-5 columns (GE Healthcare, 17-0853-01) and elution with PBS. Yields of purified V_(H) were estimated spectrophotometrically and purity was assessed using SDS PAGE.

Alternatively V_(H) were purified from the supernatants of W3110 E. coli with pJExpress vector. For this procedure up to 400 ml cultures were grown at 37° C. with 250 rpm shaking in TB media before being induced overnight with 1 mM IPTG overnight. The resulting supernatants were harvested and V_(H) purified on AKTA Pure using a Ni-Sepharose excel column (HiScale 16, GE Healthcare). Yields of purified V_(H) were estimated spectrophotometrically and purity was assessed using SDS PAGE.

b) Inhibition of Human PD-L1 and PD-L2 Binding to Recombinant Human PD-1

Purified V_(H) were serially diluted in HTRF assay buffer and tested in the HTRF PD-1:PD-L1 Inhibition assay as described previously in Example 1 and IC₅₀ values determined. These are shown in Table 3 below.

TABLE 3 HTRF PD-1:PD- Humabody ® L1 IC50 (M) 1.2 1.30E−08 1.3 2.30E−09 1.4 3.13E−09 1.5 2.66E−09 1.6 1.31E−08 1.7 4.40E−09 1.8 2.88E−08 1.9 9.13E−10 1.10 5.41E−08 1.11 5.16E−09 1.12 4.18E−09 1.13 1.70E−08 1.14 2.85E−08 1.15 3.78E−09 1.1 1.27E−09

For the PD-L2 inhibition assay, recombinant human PD-1 protein was labelled with Europium Trisbipyridine Cryptate (Cisbio cat no. 62EUSPEA) according to the manufacturer's protocol and PD-L2-Fc (Acro Biosystems cat no. PD2-H882R) was biotinylated according to EZ-link kit protocol (Thermo 21327) Serial dilutions of V_(H) were incubated with 10 nM Streptavidin AlexaFluor-647 (Life Technologies cat no. S32357), 3 nM biotinylated PD-L2-Fc and Europium Cryptate labelled PD-1-Fc (167-fold dilution) in a 10 μl assay volume for a minimum of 3 hours at room temperature. For example data see FIG. 2 and Table 4.

TABLE 4 Humabody ® IC50 (M) 1.9 7.5E−09 1.13 1.0E−08 1.5 2.2E−08 1.4 1.4E−08 1.1 4.9E−09 1.2 3.9E−08

c) Humabody® 1.1 and Humabody® 1.57 Epitope Competition Assays

Sequence optimised single domain antibodies with improved activity and/or expression levels over that of the parental (non-optimised) single domain antibodies were initially identified by testing of bacterial periplasmic extracts for their ability to compete with the binding of the parental clone 1.1 or a partially optimised Humabody® 1.57 to CHO human PD-1 cells in an FMAT epitope competition assay.

Humabody® 1.1 or 1.57 V_(H) sequence was amplified by PCR and sub-cloned into a vector enabling expression with a C terminally fused Strep tag. TG1 bacterial cultures transformed with the expression vector were cultured, periplasmic extracts prepared using extraction buffer (20% w/v sucrose, 1 mM EDTA, 50 mM Tris-HCl pH8.0) then Strep-tagged V_(H) purified from the periplasm using Strep-Tactin affinity resin (Qiagen 30002).

For the epitope competition assay reagents were prepared in FMAT assay buffer. Bacterial periplasmic extracts, buffer (total binding controls) or excess His tagged V_(H) competitor (non-specific binding control) were incubated with 1 nM 1.1Strep tagged protein or 1 nM 1.57-Strep tagged protein, 1.5 nM Strep-Tag® II monoclonal antibody (Millipore 71590), 2.5 nM Goat anti mouse Fc-Alexa Fluor 488 and 2000 CHO human PD-1 DRAQ5 stained cells per well in a 384 well black clear-bottomed assay plate. Plates were incubated for a minimum of 1.5 hours at room temperature then fluorescence measured in the FL2 (502 nm-537 nm) and FL5 (677-800 nm) channels on the Mirrorball plate reader (TTP) following excitation at 488 nm and 640 nm. Data was expressed as a 310 of the total binding control (i.e. % control) after subtraction of the background signal determined from the non-specific binding control wells. Clones that showed improved activity compared to the parent V_(H) were purified and tested multipoint in the Epitope Competition Assay for IC₅₀ determination or were tested directly in the reporter gene assay described below (data shown in Table 5).

d) Reporter Gene Assays

The ability of the V_(H) to increase functional responses in a transfected Jurkat cells as a result of PD-1:PD-L1 blockade was assessed using an NFAT-Luciferase Reporter Gene assay. A Jurkat reporter cell line expressing human PD-1 and a luciferase reporter gene under the control of a promoter with an NFAT response element and a CHO cell line expressing a T-Cell Receptor activator and human PD-L1 under the control of a tetracycline inducible promotor were generated by standard methods. Cells were prepared in bulk, then frozen and stored in liquid nitrogen.

CHO human PD-L1/TCR activator cells were thawed in a 37° C. water bath, washed once with PBS, resuspended in (Hams F12/10% FBS/1 μg/ml tetracycline) and plated at 10000 cells/well in a 96 well white TC treated assay plate. Plates were incubated at 37° C. overnight in a CO₂ incubator.

Samples were serially diluted in assay medium (RPMI+2% FBS). Jurkat PD-1 reporter cells were thawed in a 37° C. water bath, washed once with medium, then diluted into assay medium at 5e⁵ cells/ml. The media was removed from the CHO cells and 50 μl diluted sample or assay media (background control) added to the plates followed by 50 μl of the diluted Jurkat reporter cells. The plates were incubated for 6 hours at 37° C. in a CO₂ incubator, then removed from the incubator and equilibrated to room temperature for 10 mins. NanoGlo substrate (100 μl of substrate diluted 1:50 in NanGlo buffer Promega cat no. N1120) was added and the plates incubated for 10 mins at room temperature prior to measurement of luminescence signal (RLU). Data was expressed as fold/background signal. Alternatively samples were tested using the PD-1/PD-L1 Blockade Bioassay System (Promega) according to the manufacturer's protocol.

Example EC₅₀ data for activity of optimised Humabody® in the reporter assays is shown in Table 5a below.

TABLE 5a Monovalent single domain antibodies 1.1 Epitope Competition Reporter Assay Humabody ® (IC50) M EC50 (M) 1.58 4.6E−09 7.1E−09 1.59 7.8E−09 2.4E−08 1.42 3.5E−09 8.6E−09 1.43 1.3E−09 1.2E−08 1.44 1.9E−09 4.1E−08 1.45 2.5E−09 7.4E−08 1.46 1.8E−09 4.5E−08 1.47 4.4E−09 2.7E−08 1.48 1.8E−08 1.51 4.7E−09 2.3E−08 1.53 5.4E−09 1.4E−08 1.57 8.6E−09 1.62 1.4E−08 1.63 1.2E−08 1.64 2.5E−08 1.65 1.1E−08 1.67 1.2E−08 1.69 3.5E−08 1.70 2.9E−08 1.71 7.43E−09  1.72 2.61E−08  1.92  7.9E−010 1.3E−08 1.93 1.5E−09 1.91 1.4E−09 1.95 2.0E−09 1.97 1.7E−09 1.100 1.5E−09 1.98 1.1E−09 6.5E−09 1.101 3.0E−09 1.96 1.6E−09 1.94 4.2E−09 1.89 2.4E−09 1.9E−08 1.90 1.6E−09 1.99 1.4E−09 4.2E−09

TABLE 5b Multivalent single domain antibodies Multivalent Humabody ® EC₅₀ (M) 1.57-4GS-1.57 1.0E−09 1.57-6GS-1.57 8.4E−10 1.57-9GS-1.57 1.0E−09 1.57-4GS-MSA binder 1.0E−08

e) Species Cross Reactivity Testing

Purified V_(H) were tested for their ability to bind to human PD-1 (R&D Systems cat no. 1086-PD), cynomolgus PD-1 (Acro Biosystems cat no. PD-1-05254) and mouse PD-1 (R&D Systems cat no. 1021-PD) in an HTRF Binding assay format. All reagents and serially diluted V_(H) were prepared in assay buffer containing PBS, 0.1% BSA and 0.4M Potassium Fluoride. Samples or assay buffer (non-specific binding) were incubated with 2 nM human/cynomolgus or mouse PD-1, 1 nM Anti human-Fc Cryptate PAb (Cisbio cat. no. 61HFCKLB) and 30 nM anti His-D2 (CisBio cat no 61HISDLA) in black 384-shallow well assay plates for a minimum of 3 hours at room temperature. Time-resolved fluorescent emission at 620 nm and 665 nm was measured following excitation at 337 nm on the BMG PHERAstar plate reader. The HTRF ratio were calculated ((665 nm emission/620 nm emission)*10000) and the data corrected for (non-specific binding) to give the specific binding signal.

Humabody® 1.1 and variants 1.57, 1.92 showed binding to human (FIG. 3A) and cynomolgus PD-1 (FIG. 3B) recombinant protein, but did not cross react with mouse PD-1 protein (FIG. 30 ).

EC50 values are shown in Table 6 below.

TABLE 6 human PD-1 cyno PD-1 mouse PD-1 Humabody ® EC₅₀ (M) EC₅₀ (M) EC₅₀ (M) 1.1 4.0E−9  6.3E−9  No binding 1.57 5.9E−10 7.5E−10 No binding 1.92 2.6E−09 4.1E−09 No binding

Bivalent binding agents (1.57-4GS-1.57) and half-life extended binding agents (1.57-4GS-MSA binder were also tested and as with the single domain antibodies above, showed binding to human and cynomolgus PD-1 recombinant protein, but did not cross react with mouse PD-1 protein.

f) Inhibition of PD-L1 and PD-L2 Binding to CHO Human PD-1 Cells

Purified V_(H) were serially diluted in FMAT assay buffer and tested for binding to CHO human PD-1 cells as described above and for inhibition of human PD-L1 binding to CHO human PD-1 cells. The ability of purified monovalent and multivalent V_(H) to inhibit PD-L1 and PD-L2 binding to CHO human PD-1 cells respectively was confirmed using an FMAT Inhibition assay (FIGS. 4A-4C).

All reagents and serially diluted V_(H) were prepared in FMAT assay buffer. V_(H), buffer (total binding controls) or excess competitor (non-specific binding control) were incubated with 100 pM human Fc tagged human PDL-1 or 200 pM PD-L2, 3 nM anti human Fc-Alexa Fluor-488 and 2000 per well CHO human PD-1 DRAQ5 stained cells in a 384 well black clear-bottomed assay plates. Plates were incubated for 2 hours at room temperature then fluorescence measured in the FL2 (502 nm-537 nm) and FL5 (677-800 nm) channels on the Mirrorball plate reader (TTP) following excitation at 488 nm and 640 nm. Data was expressed as a % of the total binding control (i.e. % control) after subtraction of the background signal determined from the non-specific binding control wells.

IC50 values are shown below in Table 7. Example data are shown in FIGS. 4A and 4B.

TABLE 7 CHO PD-1 CHO CHO binding PD-1:PD-L1 PD-1:PD-L2 Humabody ® IC50 (M) IC50 (M) IC50 (M) 1.64  5.8E−09 9.0E−09 1.65  5.8E−09 8.5E−09 1.63  4.5E−09 6.9E−09 1.62  5.7E−09 1.0E−08 1.57  1.8E−09 2.3E−09 1.92 0.1E−09  2.8E−09 4.0E−09 1.93 2.12E−09 1.99 1.86E−09 1.91 3.56E−09 1.95 3.59E−09 1.97 3.32E−09 1.100 2.61E−09 1.98 2.08E−09 1.96 2.45E−09 1.90 3.00E−09 1.80 1.82E−09

g) Reporter Gene Assay

Functional activity of multivalent V_(H) constructs was tested in the human PD-1 reporter gene assay as described above and EC₅₀ values determined. These are shown in Table 8 below. Example data shown in FIG. 9 . Non blocking V_(H)(1) has SEQ ID NO. 587. Non-blocking V_(H)(2) has SEQ ID NO. 588. The blocking effect can be enhanced by 10 to 25 fold.

TABLE 8 Humabody ® Construct EC50 (M) 1.57-4GS-1.57 1.0E−09 1.57-6GS-1.57 8.4E−10 1.57-9GS-1.57 1.0E−09 1.57-4GS-MSA binder 1.0E−08 1.92-4GS-non-blocking VH(1) 2.0E−09 1.92-4GS-non-blockering VH(2) 1.2E−09

h) Serum Stability

Serum stability of V_(H) and multivalent V_(H) constructs was assessed by measurement of their activity following incubation for 0, 1, 4 or 7 days in mouse serum (Sigma M5905). The pre-incubated samples were serially diluted and tested in the 1.57 epitope competition assay as previously described in Example 3. Minimal loss of activity was observed following incubation with serum (FIGS. 5A-5C). Humabody® molecules tested were 1.57, 1.64, 1.57-4GS-MSA and 1.92. Example data shown in FIGS. 5A and 5B.

TABLE 9 IC 50 (M) Day 0 Day 1 Day 4 Day 7 Constructs Human Mouse Human Mouse Human Mouse Human Mouse 1.92 5.7E−09 4.4E−09 4.9E−09 4.4E−09 6.1E−09 4.7E−09 5.4E−09 5.2E−09

i) Binding of V_(H) to T-Cells

Binding of monovalent single domain antibodies and multivalent binding agents to T cells was measured using flow cytometry. Peripheral blood mononuclear cells (PBMCs) were isolated from human blood by density gradient centrifugation then CD4+ T cells purified using a negative selection isolation kit according to the manufacturer's protocol (Miltenyi Biotech cat no 130-096-533). T-cells were stimulated with 2.5 μg/ml PHA for 2-4 days in RPMI media supplemented with 10% FBS, 2 mM Glutamine, 1×Pen/Strep. Cells were transferred into 96 well plates (75000 per well), blocked for 10 mins with PBS/1% BSA, then incubated with serially diluted V_(H) in staining buffer (PBS/1% BSA) for 1 hour at 4° C. Cells were washed with staining buffer by centrifugation then incubated with 10 μg/ml biotinylated Anti His antibody for 40 mins at 4° C. Cells were washed again then stained with Streptavidin Alexa Fluor-488 (10 μg/ml) and 1:5000 diluted Live Dead near IR stain (Molecular Probes cat no. L10119) for 30 mins at 4° C. After further washing cells were fixed and fluorescence measured by flow cytometry. EC₅₀ values are shown in table 10. Example binding curve data for staining of live cell gated CD4+ T cells is shown in FIG. 6 .

TABLE 10 CD4+ T cell binding EC50 (M) Name Donor 1 Donor 2 Donor 3 Donor 4 1.92 0.8E−09 1.1E−09 1.2E−09 0.9E−09

j) V_(H) Single Domain Antibodies Demonstrate Good Stability

Purified V_(H) were subjected to size exclusion chromatography. Briefly, purified V_(H) were stored at either Na Acetate pH 5.5, 150 mM NaCl or PBS pH 7.4 for 0-14 days at either 4° C. or 40° C., and then analysed at various time points using a Waters H-Class Bio UPLC containing a PDA detector (detection at 280 nm) with separation on a Waters ACQUITY BEH 125A SEC column. Samples were injected in 10 μl volumes and were run in a mobile phase containing 200 mM NaCl, 100 mM sodium phosphate, pH 7.4+5% propan-1-ol at a flow rate of 0.4 ml/min. Data were collected for 6 minutes and the area of the monomer peak remaining after storage as compared to that present at the start (T=0) was calculated. Examples of anti-PD-1 V_(H) single domain antibodies incubated at 40° C. for 14 days are illustrated in Table 11. At incubation at 4° C. for 14 days, no significant change was seen.

TABLE 11 0 1 4 7 14 1.64 100.00 106.53 104.66 93.98 100.03 1.63 100.00 94.12 91.25 90.23 90.78 1.92 100.00 96.54 92.22 92.00 93.08

k) Effects of PD-1 Specific Humabody® on Human T Cell Activation in a Mixed Lymphocyte Reaction

Monocytes were isolated from human peripheral blood mononuclear cells (PBMCs) and differentiated into dendritic cells for 7 days using STEMCELL Technologies Dendritic Cell Differentiation media or GM-CSF and IL-4. Dendritic cells were cultured with allogeneic CD4+ T cells, isolated from PBMCs via magnetic separation. Co-cultures were incubated for 2 days in the presence of PD-1-specific Humabody® or control. T cell stimulation was measured by proliferation assay or cytokine quantification from the cell supernatant.

Humabody® 1.57 and 1.92 enhances IL-2 secretion from allogeneic dendritic cell/T cell co-culture in a concentration-dependent manner (FIG. 7A and FIG. 7B). IL-2 levels were determined after 2 or 3 days by Homogenous Time Resolved Fluorescence assay (HTRF, CisBio).

It was also shown that in the absence of stimulation by dendritic cells, there is no impact of PD-1-specific Humabody® V_(H) 1.57. This indicates that the Humabody® 1.57 is not eliciting a stimulatory signal directly so the mechanism is proposed to be blockade of the PD-1/PD-L1 inhibitory pathway.

l) Binding kinetics

Binding kinetics were measured using the methods described below

Binding kinetics to human PD-1-huFc using surface plasmon resonance

Binding kinetics of certain V_(H) single domain antibodies binding to human PD-1-huFc were measured by surface plasmon resonance (SPR) technology using Biacore T200 instrument (GE Healthcare). Recombinant human PD-1-huFc was immobilized by standard amine coupling to CM5 sensorschip (GE Healthcare) using in range of 0.1-0.01 mg/ml solution of antigen in 10 mM sodium acetate at pH 5.5. For the reference flow cell, a blank immobilisation was carried out. Single cycle kinetics assays were used to study the interaction, a five point, three-fold dilution series of each Humabody® was made with a top concentration of 30 nM. The binding kinetics were followed by flowing the Humabody® over the chip surface in HBS EP+ buffer at a flow rate of 30 μl/min. The contact time for each of the association steps was 180 seconds and the dissociation step was varied between 1200-3600 seconds. The data was fitted to a 1:1 binding model after double reference subtraction using the Biacore T200 Evaluation software. The calculated affinity and kinetic constants are shown in Table 13 below.

TABLE 13 Humabody ® ka (1/Ms) kd (1/s) KD (M) 1.1 1.09E+05 7.35E−05 6.76E−10 1.47 1.91E+05 9.48E−05 4.97E−10 1.51 1.89E+05 5.98E−05 3.17E−10 1.52 2.05E+05 6.14E−05 2.99E−10 1.52 2.97E+05 7.61E−05 2.56E−10 1.44 2.09E+05 4.18E−05 2.00E−10 1.44 2.35E+05 4.00E−05 1.70E−10 1.49 2.63E+05 2.89E−05 1.10E−10 1.48 4.96E+05 5.24E−05 1.06E−10 1.54 5.30E+05 4.53E−05 8.56E−11 1.43 4.10E+05 2.79E−05 6.81E−11 1.50 4.02E+05 2.34E−05 5.81E−11 1.41 5.37E+05 2.96E−05 5.52E−11 1.42 5.32E+05 2.69E−05 5.05E−11 1.47 7.01E+05 3.14E−05 4.48E−11 1.55 5.35E+05 1.95E−05 3.64E−11 1.56 4.32E+05 1.53E−05 3.55E−11 1.57 5.37E+05 6.68E−08 1.24E−13 1.92  6.2E+05 3.67E−05  5.9E−11

Binding Affinity and Kinetic Constant Determination Using Strep Tagged Human PD-1

Single cycle kinetics assays were used to study the interaction between human PD-1 with Humabody® on a Biacore T200 instrument (GE Healthcare). Strep tagged recombinant human PD-1 was amine coupled to one flow cell of a CM-5 chip to create a low density surface (14 RU) using standard Biacore reagents. For the reference flow cell, a blank immobilisation was carried out. A five-point, three-fold dilution series of each Humabody® was made with a top concentration of 25 nM. The binding kinetics were followed by flowing the Humabody® over the chip surface in HBS EP+ buffer at a flow rate of 30 μl/min. The contact time for each of the association steps was 180 seconds and the dissociation step was 3300 seconds. The data was fitted to a 1:1 binding model after double reference subtraction using the Biacore T200 Evaluation software. The calculated affinity and kinetic constants are shown in Table 11 below.

TABLE 14 Humabody ® ka (1/Ms) Kd (1/s) K_(D) (M) 1.57 4.05E+05 3.17E−05  7.82E−11 1.57-4GS-1.57 8.88E+05 1.54E−05  1.73E−11 1.57-6GS-1.57 7.73E+05 1.32E−05  1.71E−11 1.57-9GS-1.57 6.30E+05 1.40E−05  2.22E−11 1.84  8.3E+05 7.6E−05  9.1E−11 1.104  7.8E+05 6.3E−05  8.0E−11 1.105  9.5E+05 8.7E−05  9.1E−11 1.80 1.76E+05 5.2E−05 2.97E−11 1.77-4GS-non-blocking  5.5E+05 3.5E−05  6.3E−11 V_(H) (3) 1.96 1.52E+06 1.2E−04  8.0E−11 1.89  1.6E+06 5.6E−05 3.42E−11 Non-blocking VH(3) has SEQ ID NO. 589.

Binding Affinity and Kinetic Constant Determination Using Human PD-1-Fc

Single cycle kinetics assays were used to study the interaction between human Fc tagged human PD-1 with Humabody® on a Biacore T200 instrument (GE Healthcare). Protein G was amine coupled to two flow cells of a CM-5 chip. Human Fc tagged recombinant human PD-1 was captured onto one of the flow cells. The other was used as the reference flow cell. A five point, three-fold dilution series of each Humabody® was made with a top concentration within the range of 50 to 150 nM. The binding kinetics were followed by flowing the Humabody® over the chip surface in HBS EP+ buffer at a flow rate of 30 μl/min. The contact time for each of the association steps was 180 seconds and the dissociation step was 3600 seconds. The data was fitted to a 1:1 binding model after double reference subtraction using the Biacore T200 Evaluation software. The calculated affinity and kinetic constants are shown in Table 15 below.

TABLE 15 Humabody ® ka (1/Ms) Kd (1/s) K_(D) (M) 1.57 4.79E+5 2.11E−5 4.41E−11 1.57-9GS-1.57 1.36E+6 1.14E−7 8.40E−14 1.57-4GS-MSA 3.40E+5 1.99E−5 5.86E−11 4.35E+5 2.32E−5 5.33E−11

Example 10. Multivalent Constructs

Multivalent constructs described above were generated by linking isolated V_(H) nucleic acid sequences using a peptide linker (G4S)_(x) wherein X is 2, 4, 6 or 9 and proteins were expressed. The techniques used were based on standard molecular biology techniques.

For example, a first blocking Humabody® nucleic acid sequence was linked to a second blocking Humabody® nucleic acid sequence. In one example, the first and second sequence were the same sequences. In another example, sequence the first and second sequence were not the same sequences. Such bivalent binding agents were optionally linked to a half-life extending Humabody® nucleic acid sequence (MSA binder). Constructs were also made and expressed where monovalent V_(H) single domain antibodies were linked to a half-life extending Humabody® nucleic acid sequence (MSA binder).

Linker sequences used for these constructs are shown below in Table 16.

TABLE 16 Peptide Linker sequence Nucleic acid sequence 2GS GGGGSGGGGS GGAGGTGGAGGTTCAGGTGGAGGTGGTAGT SEQ ID No. 577 SEQ ID No. 578 4GS GGGGSGGGGSGG GGAGGTGGAGGTTCAGGAGGTGGTGGTTCT GGSGGGGS GGTGGTGGCGGTTCAGGTGGAGGTGGTAGT SEQ ID No. 579 SEQ ID No. 580 6GS GGGGSGGGGSGG GGTGGTGGCGGTTCAGGCGGAGGTGGCTCT GGSGGGGSGGGG GGAGGTGGAGGTTCAGGAGGTGGTGGTTCT SGGGGS GGCGGCGGTGGATCGGGTGGAGGTGGTAGT SEQ ID No. 581 SEQ ID No. 582 9GS GGGGSGGGGSGG GGAGGTGGAGGTTCAGGAGGTGGTGGTTCT GGSGGGGSGGGG GGTGGTGGCGGTTCAGGTGGAGGTGGTAGT SGGGGSGGGGSG GGAGGAGGTGGTTCTGGCGGAGGAGGATCG GGGSGGGGS GGTGGAGGTGGCTCAGGTGGTGGAGGTAGT SEQ ID No. 583 GGAGGCGGTGGCAGC SEQ ID No. 584

Serum stability and binding to T cells of the multivalent binding agents was assayed as described in the examples above. Data is shown above.

Example 11 In Vivo Efficacy of Humabody® Compounds in HuGEMM PD-1 Model with Subcutaneous MC38 Mouse Colon Adenocarcinoma

PD-1 HuGEMM mice are a genetically engineered mouse model (GEMM) with chimeric human/mouse PD-1 gene (h/mPD-1) containing humanized exon 2 in C57BL/6 mice. Mice were generated by Crown Biosciences and studies were run and performed by Crown.

Compounds administered were 1.57-4GS-MSA binder and Hel4. Compounds were dosed twice weekly intra peritoneal (i.p.).

MC38 mouse colon adenocarcinoma cells from Fudan IBS cell resource center (FDCC) were cultured in DMEM medium (Gibco) supplemented with 10% heat inactivated fetal bovine serum (Kang Yuan Biology) at 37° C. in an atmosphere of 5% CO₂ in air. Each mouse was inoculated subcutaneously at the right hind flank with MC38 cells (1×106) for tumor development. Totally 18 mice with desired tumor sizes were enrolled in this study. Mice were randomized into 3 experimental groups after stratification by the tumor sizes. The average tumor size of all these 18 mice was 85 mm3 at randomization. The day of randomization was denoted as day 0. The test articles were administered to the tumor-bearing mice from day 0 through day 14 with the schedule of BIW for 5 doses.

All the procedures related to animal handling, care, and the treatment in this study were performed according to guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of CrownBio following the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). At the time of routine monitoring, the animals were checked for tumor growth, mobility, food and water consumption, body weight gain/loss, eye/hair matting and any other abnormal behaviours.

Tumor size were measured twice weekly in two dimensions using a caliper, and the volume is expressed in mm3 using the formula: TV=0.5 a×b2, where a and b are the long and short diameters of the tumor, respectively. The tumor size is then used for calculation of TGI (%).

All the data is expressed as Mean±SEM. Statistical analysis of difference in tumor volume was conducted using Repeat measures two-way ANOVA. All data were analyzed using GraphPad Prism 5 and p<0.05 was considered to be statistically significant. As shown in FIG. 8 , administration of the binding agent reduced tumor volume. 

1. An isolated V_(H) single domain antibody that binds to human programmed cell death 1 protein (PD-1) comprising one of the following complementarity determining regions (CDR)1, 2 and 3 combinations: SEQ ID NOs: 1, 2, 3; SEQ ID NOs: 5, 6, 7; SEQ ID NOs: 9, 10, 11; SEQ ID NOs: 13, 14, 15; SEQ ID NOs: 17, 18, 19; SEQ ID NOs: 21, 22, 23; SEQ ID NOs: 25, 26, 27; SEQ ID NOs: 29, 30, 31; SEQ ID NOs: 33, 34, 35; SEQ ID NOs: 37, 38, 39; SEQ ID NOs: 41, 42, 43, SEQ ID NOs: 45, 46, 47; SEQ ID NOs: 49, 50, 51; SEQ ID NOs: 53, 54, 55; SEQ ID NOs: 57, 58, 59; SEQ ID NOs: 61, 62, 63; SEQ ID NOs: 65, 66, 67; SEQ ID NOs: 69, 70, 71; SEQ ID NOs: 73, 74, 75; SEQ ID NOs: 77, 78, 79; SEQ ID NOs: 101, 102, 103; SEQ ID NOs: 105, 106, 107; SEQ ID NOs: 109, 110, 111; SEQ ID NOs: 113, 114, 115; SEQ ID NOs: 117, 118, 119; SEQ ID NOs: 121, 122, 123; SEQ ID NOs: 125, 126, 127; SEQ ID NOs: 129; 130; 131; SEQ ID NOs: 133, 134, 15; SEQ ID NOs: 137, 18, 139; SEQ ID NOs: 141, 142, 143; SEQ ID NOs: 145, 146, 147; SEQ ID NOs: 149, 150, 151; SEQ ID NOs: 153, 154, 155; SEQ ID NOs: 157, 158, 159; SEQ ID NOs: 161, 162, 163; SEQ ID NOs: 165, 166, 167; SEQ ID NOs: 169, 170, 171; SEQ ID NOs: 173, 174, 175; SEQ ID NOs: 177, 178, 179; SEQ ID NOs: 181, 182, 183; SEQ ID NOs: 185, 186, 187; SEQ ID NOs: 189, 190, 191; SEQ ID NOs: 193, 194, 195; SEQ ID NOs: 197, 198, 199; SEQ ID NOs: 201, 202, 203; NOs: 205, 206, 207; SEQ ID NOs: 209, 210, 211; SEQ ID NOs: 213, 214, 215; SEQ ID NOs: 217, 218, 219, SEQ ID NOs: 221, 222, 223, SEQ ID NOs: 225, 226, 227; SEQ ID NOs: 229, 230, 231; SEQ ID NOs: 233, 234, 235; SEQ ID NOs: 237, 238, 239; SEQ ID Nos. 241, 242, 243; SEQ ID NOs: 245, 246, 247; SEQ ID NOs: 249, 250, 251; SEQ ID NOs: 253, 254, 255; SEQ ID NOs: 257, 258, 259; SEQ ID NOs: 261, 262, 263; SEQ ID NOs: 265, 266, 267; SEQ ID NOs: 269, 270, 271; SEQ ID NOs: 273, 274, 275; SEQ ID NOs: 277, 278, 279; SEQ ID NOs: 281, 282, 283; SEQ ID NOs: 285, 286, 287; SEQ ID NOs: 289, 290, 291; SEQ ID NOs: 366, 367, 368; SEQ ID NOs: 370, 371, 372; SEQ ID NOs: 374, 375, 376; SEQ ID NOs: 378, 379, 380; SEQ ID NOs: 382, 383, 384; SEQ ID NOs: 386, 387, 388; SEQ ID NOs: 390, 391, 392; SEQ ID NOs: 394, 395, 396; SEQ ID NOs: 398, 399, 400; SEQ ID NOs: 403, 403, 404; SEQ ID NOs: 406, 407, 408; SEQ ID NOs: 410, 411, 412; SEQ ID NOs: 414, 415, 416; SEQ ID NOs: 418, 419, 420; SEQ ID NOs: 422, 423, 425; SEQ ID NOs: 426, 427, 428; SEQ ID NOs: 430, 431, 432, SEQ ID NOs: 434, 436, 436; SEQ ID NOs: 438, 439, 440; SEQ ID NOs: 442, 443, 444; SEQ ID NOs: 446, 447, 448; SEQ ID NOs: 450, 451, 452; SEQ ID NOs: 454, 455, 456; SEQ ID NOs: 458, 459, 460; SEQ ID NOs: 462, 463, 464; SEQ ID NOs: 466, 467, 468; SEQ ID NOs: 470, 471, 472, SEQ ID NOs: 474, 475, 476; SEQ ID NOs: 478, 479, 480; SEQ ID NOs: 482, 483, 484; SEQ ID NOs: 486, 487, 488; SEQ ID NOs: 490, 491, 492; SEQ ID NOs: 494, 495, 496; SEQ ID NOs: 498, 499, 500; SEQ ID NOs: 502, 503, 504; SEQ ID NOs: 506, 507, 508; SEQ ID NOs: 510, 511, 512; SEQ ID NOs: 514, 515, 516; SEQ ID NOs: 518, 519, 520; SEQ ID NOs: 522, 523, 524; SEQ ID NOs: 526, 527, 528, or SEQ ID NOs: 530, 531,
 532. 2. The isolated V_(H) single domain antibody of claim comprising a sequence selected from SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 272, 276, 280, 284, 288, 292, 369, 373, 377, 381, 385, 389, 393, 397, 401, 405, 409, 413, 417 421, 425, 429, 433, 437, 441, 445, 449, 453, 457, 461, 465, 469, 473, 477, 481, 485, 489, 493, 497, 501, 505, 509, 513, 517, 521, 525, 529 or 533, or a sequence with at least 60%, 70%, 80%, 90% or 95% homology thereto.
 3. The isolated V_(H) single domain antibody of claim 1, wherein said single domain antibody is conjugated to a toxin, enzyme, radioisotope, half-life extending moiety, label, therapeutic molecule or other chemical moiety, optionally wherein said half-life extending moiety is selected from the group consisting of an albumin binding moiety, a transferrin binding moiety, a polyethylene glycol molecule, a recombinant polyethylene glycol molecule, human serum albumin, a fragment of human serum albumin, and an albumin binding peptide or single domain antibody that binds to human serum albumin.
 4. (canceled)
 5. The isolated V_(H) single domain antibody of claim 1 obtained or obtainable from a transgenic rodent that expresses a transgene comprising human V, D and J regions, optionally wherein said rodent does not produce any functional endogenous light and heavy chains.
 6. (canceled)
 7. An isolated binding agent that binds to essentially the same epitope as the single domain antibody of claim
 1. 8. An isolated binding agent that competes for binding to human PD-1 with the single domain antibody of claim
 1. 9. The binding agent of claim 8, wherein said binding agent is an antibody or fragment thereof.
 10. The isolated binding agent of claim 9, wherein said fragment is a Fab, Fab′, F(ab′)2, dAb, Fd, Fv, a single chain Fv fragment, a human heavy chain variable domain (VH), or an isolated CDR.
 11. An isolated binding agent comprising the single domain antibody of claim
 1. 12. The isolated binding agent of claim 7 wherein said single domain antibody is linked to a second binding molecule that does not bind to PD-1, optionally wherein said second single domain antibody binds to an immuno-oncology target, or wherein said single domain antibody is linked to a second binding molecule that binds to PD-1 at the same, overlapping or different epitope. 13-14. (canceled)
 15. The isolated binding agent of claim 7, wherein said binding agent is an antibody or a fragment thereof, optionally wherein said binding molecule is a human heavy chain variable domain (V_(H)). 16-19. (canceled)
 20. An immunoconjugate comprising the single domain antibody of claim 1 linked to a therapeutic agent, optionally wherein said therapeutic agent is a toxin, enzyme, radioisotope or other chemical moiety.
 21. (canceled)
 22. A pharmaceutical composition comprising the single domain antibody of claim 1 and a pharmaceutical carrier.
 23. A method for treating a cancer, an immune disorder, neurological disease, inflammatory disorder, allergy, transplant rejection, viral infection, immune deficiency or other immune system-related disorder, or a method of modulating an immune response, comprising administering a therapeutically effective amount of the single domain antibody of claim
 1. 24-26. (canceled)
 27. The method of claim 23, wherein said cancer is selected from bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, breast cancer, brain cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, kidney cancer, sarcoma of soft tissue, cancer of the urethra, cancer of the bladder, renal cancer, lung cancer, non-small cell lung cancer, thymoma, urothelial carcinoma leukemia, prostate cancer, mesothelioma, adrenocortical carcinoma, lymphomas, such as such as Hodgkin's disease, non-Hodgkin's, gastric cancer, and multiple myelomas.
 28. (canceled)
 29. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the single domain antibody of claim 1, optionally wherein the isolated nucleic acid molecule is selected from SEQ ID NOs: 293 to 365 or 534 to
 575. 30. (canceled)
 31. A vector comprising the nucleic acid of claim
 29. 32. A host cell comprising the vector of claim 31, optionally wherein said host cell is a bacterial, yeast, insect, plant, viral or mammalian cell.
 33. (canceled)
 34. A method for producing said single domain antibody of claim 1, comprising expressing a nucleic acid encoding said single domain antibody in a host cell and isolating the single domain antibody from the host cell.
 35. A kit comprising the single domain antibody of claim
 1. 36. A method for detecting the presence of human PD-1 in a test sample comprising contacting said sample with the single domain antibody of claim 1 and at least one detectable label and detecting binding of said single domain antibody to human PD-1.
 37. A method for producing a single V_(H) domain antibody that binds to human PD-1 comprising a) immunising a transgenic animal that expresses a nucleic acid construct comprising human heavy chain V genes and that is not capable of making functional endogenous light or heavy chains with an PD-1 antigen, b) generating a library from said animal c) isolating single V_(H) domain antibodies from said libraries and d) identifying a single V_(H) domain antibody that binds to human PD-1 and e) isolating said antibody. 38-39. (canceled)
 40. A transgenic rodent that produces a heavy chain only antibody that binds to human PD-1, wherein the transgenic rodent expresses a nucleic acid construct comprising human heavy chain V genes, is not capable of making functional endogenous light or heavy chains, and has been immunized with an PD-1 antigen.
 41. (canceled)
 42. A bispecific molecule comprising the single domain antibody of claim 1 linked to a second functional moiety having a different binding specificity than said single domain antibody, optionally wherein said second moiety is an antibody, an antibody fragment or antibody mimetic, optionally wherein said second moiety binds to an immunooncology target. 43-44. (canceled) 